Different modes of Notch activation and strength regulation in the spermathecal secretory lineage.

The strength of Notch signaling contributes to pleiotropic actions of Notch; however, we do not yet have a full understanding of the molecular regulation of Notch-signaling strength. We have investigated the mode of Notch activation in binary fate specification in the Drosophila spermathecal linage, where Notch is asymmetrically activated across three divisions to specify different cell fates. Using clonal analysis, we show that Delta (Dl) serves as the ligand for Notch in the first and second divisions. Dl and Serrate (Ser) function redundantly in the third division. Compared with the third division, cell-fate decision in the second division requires a lower level of Suppressor of Hairless protein, and, consequently, a lower level of Notch signaling. Several Notch endosomal trafficking regulators differentially regulate Notch signaling between the second and third divisions. Here, we demonstrate that cell differentiation in spermathecae involves different Notch-activation modes, Notch-signaling strengths and Notch-trafficking regulations. Thus, the Drosophila spermathecal lineage is an exciting model for probing the molecular mechanisms that modulate the Notch signaling pathway.

Loss of Capicua alters early T cell development and predisposes mice to T cell lymphoblastic leukemia/lymphoma.

Capicua (CIC) regulates a transcriptional network downstream of the RAS/MAPK signaling cascade. In Drosophila, CIC is important for many developmental processes, including embryonic patterning and specification of wing veins. In humans, CIC has been implicated in neurological diseases, including spinocerebellar ataxia type 1 (SCA1) and a neurodevelopmental syndrome. Additionally, we and others have reported mutations in CIC in several cancers. However, whether CIC is a tumor suppressor remains to be formally tested. In this study, we found that deletion of Cic in adult mice causes T cell acute lymphoblastic leukemia/lymphoma (T-ALL). Using hematopoietic-specific deletion and bone marrow transplantation studies, we show that loss of Cic from hematopoietic cells is sufficient to drive T-ALL. Cic-null tumors show up-regulation of the KRAS pathway as well as activation of the NOTCH1 and MYC transcriptional programs. In sum, we demonstrate that loss of CIC causes T-ALL, establishing it as a tumor suppressor for lymphoid malignancies. Moreover, we show that mouse models lacking CIC in the hematopoietic system are robust models for studying the role of RAS signaling as well as NOTCH1 and MYC transcriptional programs in T-ALL.

A high Notch pathway activation predicts response to γ secretase inhibitors in proneural subtype of glioma tumor-initiating cells.

Genomic, transcriptional, and proteomic analyses of brain tumors reveal subtypes that differ in pathway activity, progression, and response to therapy. However, a number of small molecule inhibitors under development vary in strength of subset and pathway-specificity, with molecularly targeted experimental agents tending toward stronger specificity. The Notch signaling pathway is an evolutionarily conserved pathway that plays an important role in multiple cellular and developmental processes. We investigated the effects of Notch pathway inhibition in glioma tumor-initiating cell (GIC, hereafter GIC) populations using γ secretase inhibitors. Drug cytotoxicity testing of 16 GICs showed differential growth responses to the inhibitors, stratifying GICs into responders and nonresponders. Responder GICs had an enriched proneural gene signature in comparison to nonresponders. Also gene set enrichment analysis revealed 17 genes set representing active Notch signaling components NOTCH1, NOTCH3, HES1, MAML1, DLL-3, JAG2, and so on, enriched in responder group. Analysis of The Cancer Genome Atlas expression dataset identified a group (43.9%) of tumors with proneural signature showing high Notch pathway activation suggesting γ secretase inhibitors might be of potential value to treat that particular group of proneural glioblastoma (GBM). Inhibition of Notch pathway by γ secretase inhibitor treatment attenuated proliferation and self-renewal of responder GICs and induces both neuronal and astrocytic differentiation. In vivo evaluation demonstrated prolongation of median survival in an intracranial mouse model. Our results suggest that proneural GBM characterized by high Notch pathway activation may exhibit greater sensitivity to γ secretase inhibitor treatment, holding a promise to improve the efficiency of current glioma therapy.

Strong Notch activation hinders bevacizumab efficacy in advanced colorectal cancer.

AIM: To assess the role of Notch activation in predicting bevacizumab efficacy in colorectal cancer (CRC). MATERIALS & METHODS: Notch activation was evaluated by immunohistochemistry (IHC) on 65 CRC enrolled within randomized clinical trials assessing first-line bevacizumab-based chemotherapy and on 21 CRC treated with chemotherapy alone. RESULTS: Strong Notch (IHC 3+) activation was negatively associated with response (18 vs 62% in low Notch cases [IHC 0, 1, 2+]; p = 0.016), progression-free survival (4.9 vs 12.1 months; p = 0.002) and overall survival (19.3 vs 30.4 months; p = 0.039). No correlation was found between Notch activation and clinical outcome in CRC treated with chemotherapy alone. CONCLUSION: A potential role of Notch activation in the antitumor activity of bevacizumab could be hypothesized.

Apical-basal polarity precisely determines intestinal stem cell number by regulating Prospero threshold.

Apical-basal polarity and cell-fate determinants are crucial for the cell fate and control of stem cell numbers. However, their interplay leading to a precise stem cell number remains unclear. Drosophila pupal intestinal stem cells (pISCs) asymmetrically divide, generating one apical ISC progenitor and one basal Prospero (Pros)+ enteroendocrine mother cell (EMC), followed by symmetric divisions of each daughter before adulthood, providing an ideal system to investigate the outcomes of polarity loss. Using lineage tracing and ex vivo live imaging, we identify an interlocked polarity regulation network precisely determining ISC number: Bazooka inhibits Pros accumulation by activating Notch signaling to maintain stem cell fate in pISC apical daughters. A threshold of Pros promotes differentiation to EMCs and avoids ISC-like cell fate, and over-threshold of Pros inhibits miranda expression to ensure symmetric divisions in pISC basal daughters. Our work suggests that a polarity-dependent threshold of a differentiation factor precisely controls stem cell number.

High-Throughput Analysis to Identify Activators of Notch Signaling.

The Notch pathway regulates many cellular functions in a context-dependent manner. Depending on the cell type, either the activation or inhibition of Notch signaling can influence many processes such as cellular proliferation, specification, differentiation, and survival. The activation of Notch signaling has been shown to have therapeutic advantages in some cancers, thus having a method to identify Notch-activating compounds is needed. In this chapter we outline a method for high-throughput analysis of potential Notch pathway activators in a pancreatic neuroendocrine tumor cell line as an example. We also include the steps for subsequent validation of results and preclinical testing.

Mammalian NOTCH Receptor Activation and Signaling Protocols.

The NOTCH signaling pathway is one of the highly conserved key pathways involved in most cell differentiation and proliferation processes during both developmental and adult stages in most animals. The NOTCH signaling pathway appears to be very simple but the existence of several receptors and ligands, their posttranslational modifications, their activation in the cell surface and its migration to the cell nucleus, as well as their interaction with multiple signaling pathways in the cytoplasm and the nucleus of cells, make the study of its function very complex.To determine the activation of NOTCH signaling in animal cells, several complementary approaches can be performed. One of them is the analysis of the transcription of NOTCH receptor target genes HES/HEY by qRT-PCR and Western blot. This approach would give us an idea of the global NOTCH activation and signaling. We can also analyze the NOTCH transcriptional activity by luciferase assays to determine the global or specific activation of NOTCH receptors under a given treatment or in response to the modification of gene expression. On the other hand, we can determine the specific activation of each NOTCH receptor by Western blot with antibodies that recognize the active forms of each NOTCH receptor. For this assay will be very important to collect the cells to be analyzed under the appropriate conditions. Finally, we can detect the intracellular domain of each NOTCH receptor into the cell nucleus by confocal microscopy using the appropriate antibodies that recognize the intracellular domain of the receptors.

Developmental Perspectives on Arterial Fate Specification.

Blood vessel acquisition of arterial or venous fate is an adaptive phenomenon in response to increasing blood circulation during vascular morphogenesis. The past two decades of effort in this field led to development of a widely accepted paradigm of molecular regulators centering on VEGF and Notch signaling. More recent findings focused on shear stress-induced cell cycle arrest as a prerequisite for arterial specification substantially modify this traditional understanding. This review aims to summarize key molecular mechanisms that work in concert to drive the acquisition of arterial fate in two distinct developmental settings of vascular morphogenesis: de novo vasculogenesis of the dorsal aorta and postnatal retinal angiogenesis. We will also discuss the questions and conceptual controversies that potentially point to novel directions of investigation and possible clinical relevance.

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1.

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.