Notch Effector Research Articles

Rapid population-wide declines in stem cell number and activity during reproductive aging in C. elegans.

C. elegans hermaphrodites display dramatic age-related decline of reproduction early in life, while somatic functions are still robust. To understand reproductive aging, we analyzed the assembly line of oocyte production that generates fertilized eggs. Aging germlines displayed both sporadic and population-wide changes. A small fraction of aging animals displayed endomitotic oocytes in the germline and other defects. By contrast, all animals displayed age-related decreases in germline size and function. As early as day 3 of adulthood, animals displayed fewer stem cells and a slower cell cycle, which combine to substantially decrease progenitor zone output. The C. elegans germline is the only adult tissue that contains stem cells, allowing the analysis of stem cells in aging. To investigate the mechanism of the decrease in stem cell number, we analyzed the Notch signaling pathway. The Notch effectors LST-1 and SYGL-1 displayed age-related decreases in expression domains, suggesting a role for Notch signaling in germline aging. The results indicate that although sporadic defects account for the sterility of some animals, population-wide changes account for the overall pattern of reproductive aging.

Myocardial Notch1-Rbpj deletion does not affect NOTCH signaling, heart development or function.

During vertebrate cardiac development NOTCH signaling activity in the endocardium is essential for the crosstalk between endocardium and myocardium that initiates ventricular trabeculation and valve primordium formation. This crosstalk leads later to the maturation and compaction of the ventricular chambers and the morphogenesis of the cardiac valves, and its alteration may lead to disease. Although endocardial NOTCH signaling has been shown to be crucial for heart development, its physiological role in the myocardium has not been clearly established. Here we have used mouse genetics to evaluate the role of NOTCH in myocardial development. We have inactivated the unique and ubiquitous NOTCH effector RBPJ in early cardiomyocytes progenitors, and examined its consequences in cardiac development and function. Our results show that mice with Tnnt2-Cre-mediated myocardial-specific deletion of Rbpj develop to term, with homozygous mutant animals showing normal expression of cardiac development markers, and normal adult heart function. Similar observations have been obtained after Notch1 deletion with Tnnt2-Cre. We have also deleted Rbpj in both myocardial and endocardial progenitor cells, using the Nkx2.5-Cre driver, resulting in ventricular septal defect (VSD), double outlet right ventricle (DORV), and bicuspid aortic valve (BAV), due to NOTCH signaling abrogation in the endocardium of cardiac valves. Our data demonstrate that NOTCH-RBPJ inactivation in the myocardium does not affect heart development or adult cardiac function.

Notch Signaling Regulates Mitochondrial Metabolism and NF-κB Activity in Triple-Negative Breast Cancer Cells via IKKα-Dependent Non-canonical Pathways.

Triple negative breast cancer (TNBC) patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Notch signaling is a key pathway regulating TNBC CSC survival. Treatment of TNBC with PI3K or mTORC1/2 inhibitors results in drug-resistant, Notch-dependent CSC. However, downstream mechanisms and potentially druggable Notch effectors in TNBC CSCs are largely unknown. We studied the role of the AKT pathway and mitochondrial metabolism downstream of Notch signaling in TNBC CSC from cell lines representative of different TNBC molecular subtypes as well as a novel patient-derived model. We demonstrate that exposure of TNBC cells to recombinant Notch ligand Jagged1 leads to rapid AKT phosphorylation in a Notch1-dependent but RBP-Jκ independent fashion. This requires mTOR and IKKα. Jagged1 also stimulates mitochondrial respiration and fermentation in an AKT- and IKK-dependent fashion. Notch1 co-localizes with mitochondria in TNBC cells. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor PF-03084014 in combination with AKT inhibitor MK-2206 or IKK-targeted NF-κB inhibitor Bay11-7082 blocks secondary mammosphere formation from sorted CD90hi or CD44+CD24low (CSCs) cells. A TNBC patient-derived model gave comparable results. Besides mitochondrial oxidative metabolism, Jagged1 also triggers nuclear, NF-κB-dependent transcription of anti-apoptotic gene cIAP-2. This requires recruitment of Notch1, IKKα and NF-κB to the cIAP-2 promoter. Our observations support a model where Jagged1 triggers IKKα-dependent, mitochondrial and nuclear Notch1 signals that stimulate AKT phosphorylation, oxidative metabolism and transcription of survival genes in PTEN wild-type TNBC cells. These data suggest that combination treatments targeting the intersection of the Notch, AKT and NF-κB pathways have potential therapeutic applications against CSCs in TNBC cases with Notch1 and wild-type PTEN expression.

Therapeutic Implications of Targeting Notch Pathway in Glioma

The purpose of this study is to evaluate the effects of targeting Notch pathway on the glioma pathobiology. Gliomas are the most common cancer of central nervous system (CNS) and are highly invasive and vascularized. Given the critical role of Notch pathway in gliogenesis and maintenance of neural progenitors, its pivotal role in CNS carcinogenesis is also substantiated. Additionally, the products of Notch pathway are also known to modulate hypoxia and angiogenesis, which are well-known hallmarks of glioma. Therefore, we sought to investigate how Notch pathway and its interactome can drive the gliomagenesis, and the therapeutic insinuations associated with targeting specific component(s) of the pathway. We used specific γ-secretase inhibitors to block the cleavage of Notch Intracellular domain and the subsequent activation of the Notch signaling. This was followed by the assessment of the downstream effects on cell proliferation and the targeting of Notch effector proteins and associated pathways. Additionally, we probed the synergistic effects of Notch inhibition with radiation treatment (RT), and in combination with Temozolomide. Lastly, we used RNAi to target the Notch receptors individually, in order to probe the effects on glioma pathogenesis and evaluate their therapeutic potential. We observed a significant decline in cell proliferation after treatment with γ-secretase inhibitors, and in combination with RT and temozolomide. In addition, we observed a comprehensive targeting of Notch pathway, both at canonical and non-canonical levels. All these phenotypic effects were augmented with a synergy of these inhibitors with both RT and temozolomide. Lastly, we observed context-specific effects of targeting Notch receptors on cell proliferation, invasion, EMT, and their cross-talk with other radioresistance pathways. Targeting notch pathway modulates the vital cellular processes that are implicated in the tumorigenesis of the gliomas. All these endeavors may lead to better treatment outcomes for the patients. Further studies to authenticate the druggability of notch effectors and their interacting partners should be investigated to determine if this combinatorial targeting could be used to design personalized therapeutic interventions for GBM patients.

PO-152 Differentiation of lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) by gene co-expression analysis of notch signalling targets

IntroductionLung carcinomas are very aggressive malignancies, which remain leading cancer-related cause of death among men and women worldwide. Regarding histological classification LUSC and LUAD account for the majority of lung tumours among non-small cell carcinomas (NSCLC). Notch signalling is evolutionarily-conserved pathway that regulates many cellular processes such as proliferation, differentiation and epithelial-to-mesenchymal transition (EMT). To date, aberrant Notch signalling has been linked with wide variety of malignancies, including lung tumours. However the association between co-expression profile of Notch downstream effector and lung cancer subtypes remains unclear. Therefore the aim of our study was to investigate functional gene co-expression networks to search for candidate biomarkers or therapeutic targets.Material and methodsIn our analysis we used RNASeq expression and clinical data downloaded from The Cancer Genome Atlas (TGCA). Gene Set Enrichment Analysis (GSEA) performed for canonical pathways pointed to Notch pathway as one of the most significant. Subsequently we analysed expression of downstream Notch effectors, 2949 HES/HEY transcription factors targets. To this extent used Weighted Gene Co-expression Network Analysis (WGCNA) to find differences in gene expression profile between LUSC and LUAD.Results and discussionsThe analysis of Notch pathway downstream targets which expression is regulated by HES/HEY transcription factors, identified 9 gene modules highly correlated with cancer type, with two of them as the most promising. Functional analysis revealed that among the differentially expressed genes were those involved in proliferation, cell cycle regulation, DNA repair, EMT, adhesion and metabolic processes, for example: TP63, PIK3CA, ADAM23, DLG1, FXR1, SENP5, TTK, BIRC5, KIF18A, KIF14, KIF4A, MCM4, MCM10. For one module the highly connected hub gene is TP63, acts as oncogene in many tumour types including squamous cell carcinomas, and for the second module is KIF4A. Interestingly, all listed genes were found to be overexpressed in LUSC and downexpressed in LUAD.ConclusionOur analysis could be valuable for better understanding of the molecular mechanism of lung carcinoma as well as Notch signalling in lung cancer with emphasis of pathway gene expression as useful biomarker for differentiating cancer progression in lung cancer subtypes.AcknowledgmentThis study was founded by the National Science Centre, Poland nr 2016/23/N/NZ2/02575 and Medical University of Lodz 503/0-078-02/503-01-004

Gremlin activates the Notch pathway linked to renal inflammation.

Preclinical studies suggest that Gremlin participates in renal damage and could be a potential therapeutic target for human chronic kidney diseases. Inflammation is a common characteristic of progressive renal disease, and therefore novel anti-inflammatory therapeutic targets should be investigated. The Notch signaling pathway is involved in kidney development and is activated in human chronic kidney disease, but whether Gremlin regulates the Notch pathway has not been investigated. In cultured tubular cells, Gremlin up-regulated gene expression of several Notch pathway components, increased the production of the canonical ligand Jagged-1, and caused the nuclear translocation of active Notch-1 (N1ICD). In vivo administration of Gremlin into murine kidneys elicited Jagged-1 production, increased N1ICD nuclear levels, and up-regulated the gene expression of the Notch effectors hes-1 and hey-1 All these data clearly demonstrate that Gremlin activates the Notch pathway in the kidney. Notch inhibition using the γ-secretase inhibitor DAPT impaired renal inflammatory cell infiltration and proinflammatory cytokines overexpression in Gremlin-injected mice and in experimental models of renal injury. Moreover, Notch inhibition blocked Gremlin-induced activation of the canonical and noncanonical nuclear factor-κB (NF-κB) pathway, identifying an important mechanism involved in the anti-inflammatory actions of Notch inhibition. In conclusion, Gremlin activates the Notch pathway in the kidney and this is linked to NF-κB-mediated inflammation, supporting the hypothesis that Notch inhibition could be a potential anti-inflammatory strategy for renal diseases.

PO-151 Notch signalling differentiates disease-free survival in prostate cancer patients by affecting the epithelial-to-mesenchymal transition-associated processes

Introduction In majority of cases tumour of the prostate is developing with benign age-related prostatic hyperplasia. However some of them are very aggressive. Therefore, the dispute how to differentiate patients with malignant vs benign form of the disease and patients, which have risk of recurrence remains open. Notch signalling plays a pivotal role in prostate homeostasis and its deregulation is associated with prostate tumour recurrence through involvement in epithelial-to-mesenchymal transition (EMT). This study aimed to determine role of Notch signalling and its downstream effects in recurrence of prostate cancer and to reveal network of genes that differential expression may be substantially significant for relapse. Material and methods TCGA PRAD cohort was examined regarding expression of Notch receptors and their association with tumour recurrence. DFS analysis was performed using EvaluateCutpoints (log-rank, p Results and discussions DFS analysis showed common profile for all Notch receptors with low expression as favourable prognosis. GSEA analyses between favourable/unfavourable expression groups resulted in overrepresentation of gene sets directly involved in EMT (adhesion, integrins, WNT signalling) in unfavourable group. MFA performed on EMT markers showed that their expression clearly differentiates disease relapse from tumour free patients. Finally, network of downstream Notch effectors revealed 745 disease recurrence related genes involved mainly in carcinogenesis-associated processes (cell cycle, apoptosis) and adhesion supporting our primary hypothesis. Conclusion This research revealed the role of Notch in prostate cancer recurrence mechanism. It showed Notch receptors as potential markers indicating risk of disease recurrence. Moreover, downstream Notch signalling tends to affect cell cycle, tissue remodelling and architecture, thus indicating potential to trigger EMT. Acknowledgments This study was funded by the National Science Centre, Poland 2016/23/N/NZ2/02372 and Medical University of Lodz 503/0-078-02/503-01-004.

Reconstitution of an Ultradian Oscillator in Mammalian Cells by a Synthetic Biology Approach.

The Notch effector gene Hes1 is an ultradian clock exhibiting cyclic gene expression in several progenitor cells, with a period of a few hours. Because of the complexity of studying Hes1 in the endogenous setting, and the difficulty of imaging these fast oscillations in vivo, the mechanism driving oscillations has never been proven. Here, we applied a "build it to understand it" synthetic biology approach to construct simplified "hybrid" versions of the Hes1 ultradian oscillator combining synthetic and natural parts. We successfully constructed a simplified synthetic version of the Hes1 promoter matching the endogenous regulation logic. By mathematical modeling and single-cell real-time imaging, we were able to demonstrate that Hes1 is indeed able to generate stable oscillations by a delayed negative feedback loop. Moreover, we proved that introns in Hes1 contribute to the transcriptional delay but may not be strictly necessary for oscillations to occur. We also developed a novel reporter of endogenous Hes1 oscillations able to amplify the bioluminescence signal 5-fold. Our results have implications also for other ultradian oscillators.

TBBPA and Its Alternatives Disturb the Early Stages of Neural Development by Interfering with the NOTCH and WNT Pathways.

Tetrabromobisphenol A (TBBPA), as well as its alternatives Tetrabromobisphenol S (TBBPS) and Tetrachlorobisphenol A (TCBPA), are widely used halogenated flame retardants. Their high detection rates in human breast milk and umbilical cord serum have raised wide concerns about their adverse effects on human fetal development. In this study, we evaluated the cytotoxicity and neural developmental toxicity of TBBPA, TBBPS, and TCBPA with a mouse embryonic stem cell (mESC) system, at human body fluid and environmental relevant doses. All the three compounds showed similar trends in their cytotoxic effects. However, while TBBPA and TBBPS stimulated ESC neural differentiation, TCBPA significantly inhibited neurogenesis. Mechanistically, we demonstrated that, as far as the NOTCH (positive regulator) and WNT (negative regulator) pathways were concerned, TBBPA only partially and slightly disturbed them, whereas TBBPS significantly inhibited the WNT pathway, and TCBPA down-regulated the expression of NOTCH effectors but increased the WNT signaling, actions which both inhibited neural specification. In conclusion, our findings suggest that TBBPS and TCBPA may not be safe alternatives to TBBPA, and their toxicity need to be comprehensively evaluated.

Neural Crest Derived Cardiomyocytes Regulate Cardiac Trabeculation and Adult Heart Function.

The cardiac neural crest arise from the multi‐potent, migratory neural crest cell population and contribute to cardiovascular development, including contributions to cardiac endothelium and myocardium, formation of the great vessels and their valves, as well as septation of the chambers. However, very little is known about the neural crest contribution to the myocardium. This study aims to selectively decipher the function of the neural crest derived cardiomyocytes (NC‐Cms) without the confounding roles of the contributions of neural crest to other tissues, including endocardium. Zebrafish provide a unique genetic model to study this population because of their developmental transparency and they have a relatively large neural crest contribution of NC‐Cms compared to mammals. By creating Zebrafish transgenic lines that both lineage label and allow for ablation of only NC‐Cms, we find that the embryonic NC‐Cms arrive early in zebrafish heart development, makeup ~10% of the embryonic cardiomyocyte population and are steady state by two days post fertilization (2dpf). A majority of this contribution is to the ventricle in a discrete pattern around the apex of the ventricle. To test the function of NC‐Cms during heart development, we genetically ablated them during their early stages (up to 2dpf) and then observed heart development and phenotype up to 14dpf and into adulthood. Surprisingly embryonic ablation of the NC‐Cms was largely dispensable for heart development with a non‐lethal but significant defect in patterning of ventricle trabeculation. Importantly, embryonic ablation of NC‐Cms yielded adults with hypertrabeculated hearts and impaired performance in cardiac stress tests. We investigated the relation of the NC‐Cms to known Notch and Neuregulin effectors of embryonic trabeculation and speculate on their mechanistic regulation of this pathway. These results present a new model of human left ventricular non‐compaction or hypertrabeculation cardiomyopathy, and adult heart failure. Importantly these results demonstrate how a subtle and relatively minor developmental cell population can have detrimental effects on adult cardiac function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oscillatory Control of Notch Signaling in Development.

The Notch effectors Hes1 and Hes7 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner during neurogenesis and somitogenesis. These two biological events exhibit different types of oscillations: anti-/out-of-phase oscillation in neural stem cells during neurogenesis and in-phase oscillation in presomitic mesoderm (PSM) cells during somitogenesis. Accelerated or delayed Dll1 expression by shortening or elongating the size of the Dll1 gene, respectively, dampens or quenches Dll1 oscillation at intermediate levels, a phenomenon known as "amplitude/oscillation death" of coupled oscillators. Under this condition, both Hes1 oscillation in neural stem cells and Hes7 oscillation in PSM cells are also dampened. As a result, maintenance of neural stem cells is impaired, leading to microcephaly, while somite segmentation is impaired, leading to severe fusion of somites and their derivatives, such as vertebrae and ribs. Thus, the appropriate timing of Dll1 expression is critical for the oscillatory expression in Notch signaling and normal processes of neurogenesis and somitogenesis. Optogenetic analysis indicated that Dll1 oscillations transfer the oscillatory information between neighboring cells, which may induce anti-/out-of-phase and in-phase oscillations depending on the delay in signaling transmission. These oscillatory dynamics can be described in a unified manner by mathematical modeling.

Notch pathway signaling in the skin antagonizes Merkel cell development

Merkel cells are mechanosensitive skin cells derived from the epidermal lineage whose development requires expression of the basic helix-loop-helix transcription factor Atoh1. The genes and pathways involved in regulating Merkel cell development during embryogenesis are poorly understood. Notch pathway signaling antagonizes Atoh1 expression in many developing body regions, so we hypothesized that Notch signaling might inhibit Merkel cell development. We found that conditional, constitutive overexpression of the Notch intracellular domain (NICD) in mouse epidermis significantly decreased Merkel cell numbers in whisker follicles and touch domes of hairy skin. Conversely, conditional deletion of the obligate NICD binding partner RBPj in the epidermis significantly increased Merkel cell numbers in whisker follicles, led to the development of ectopic Merkel cells outside of touch domes in hairy skin epidermis, and altered the distribution of Merkel cells in touch domes. Deletion of the downstream Notch effector gene Hes1 also significantly increased Merkel cell numbers in whisker follicles. Together, these data demonstrate that Notch signaling regulates Merkel cell production and patterning.

Brain endothelial cells induce astrocytic expression of the glutamate transporter GLT-1 by a Notch-dependent mechanism.

Neuron-secreted factors induce astrocytic expression of the glutamate transporter, GLT-1 (excitatory amino acid transporter 2). In addition to their elaborate anatomic relationships with neurons, astrocytes also have processes that extend to and envelop the vasculature. Although previous studies have demonstrated that brain endothelia contribute to astrocyte differentiation and maturation, the effects of brain endothelia on astrocytic expression of GLT-1 have not been examined. In this study, we tested the hypothesis that endothelia induce expression of GLT-1 by co-culturing astrocytes from mice that utilize non-coding elements of the GLT-1 gene to control expression of reporter proteins with the mouse endothelial cell line, bEND.3. We found that endothelia increased steady state levels of reporter and GLT-1 mRNA/protein. Co-culturing with primary rat brain endothelia also increases reporter protein, GLT-1 protein, and GLT-1-mediated glutamate uptake. The Janus kinase/signal transducer and activator of transcription 3, bone morphogenic protein/transforming growth factor β, and nitric oxide pathways have been implicated in endothelia-to-astrocyte signaling; we provide multiple lines of evidence that none of these pathways mediate the effects of endothelia on astrocytic GLT-1 expression. Using transwells with a semi-permeable membrane, we demonstrate that the effects of the bEND.3 cell line are dependent upon contact. Notch has also been implicated in endothelia-astrocyte signaling invitro and invivo. The first step of Notch signaling requires cleavage of Notch intracellular domain by γ-secretase. We demonstrate that the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester blocks endothelia-induced increases in GLT-1. We show that the levels of Notch intracellular domain are higher in nuclei of astrocytes co-cultured with endothelia, an effect also blocked by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Finally, infection of co-cultures with shRNA directed against recombination signal binding protein for immunoglobulin kappa J, a Notch effector, also reduces endothelia-dependent increases in enhanced green fluorescent protein and GLT-1. Together, these studies support a novel role for Notch in endothelia-dependent induction of GLT-1 expression. Cover Image for this issue: doi. 10.1111/jnc.13825.

MiR-7 Buffers Differentiation in the Developing Drosophila Visual System

SummaryThe 40,000 neurons of the medulla, the largest visual processing center of the Drosophila brain, derive from a sheet of neuroepithelial cells. During larval development, a wave of differentiation sweeps across the neuroepithelium, converting neuroepithelial cells into neuroblasts that sequentially express transcription factors specifying different neuronal cell fates. The switch from neuroepithelial cells to neuroblasts is controlled by a complex gene regulatory network and is marked by the expression of the proneural gene l’sc. We discovered that microRNA miR-7 is expressed at the transition between neuroepithelial cells and neuroblasts. We showed that miR-7 promotes neuroepithelial cell-to-neuroblast transition by targeting downstream Notch effectors to limit Notch signaling. miR-7 acts as a buffer to ensure that a precise and stereotypical pattern of transition is maintained, even under conditions of environmental stress, echoing the role that miR-7 plays in the eye imaginal disc. This common mechanism reflects the importance of robust visual system development.

Disruption of spatiotemporal hypoxic signaling causes congenital heart disease in mice.

Congenital heart disease (CHD) represents the most prevalent inborn anomaly. Only a minority of CHD cases are attributed to genetic causes, suggesting a major role of environmental factors. Nonphysiological hypoxia during early pregnancy induces CHD, but the underlying reasons are unknown. Here, we have demonstrated that cells in the mouse heart tube are hypoxic, while cardiac progenitor cells (CPCs) expressing islet 1 (ISL1) in the secondary heart field (SHF) are normoxic. In ISL1+ CPCs, induction of hypoxic responses caused CHD by repressing Isl1 and activating NK2 homeobox 5 (Nkx2.5), resulting in decreased cell proliferation and enhanced cardiomyocyte specification. We found that HIF1α formed a complex with the Notch effector hes family bHLH transcription factor 1 (HES1) and the protein deacetylase sirtuin 1 (SIRT1) at the Isl1 gene. This complex repressed Isl1 in the hypoxic heart tube or following induction of ectopic hypoxic responses. Subsequently, reduced Isl1 expression abrogated ISL1-dependent recruitment of histone deacetylases HDAC1/5, inhibiting Nkx2.5 expression. Inactivation of Sirt1 in ISL1+ CPCs blocked Isl1 suppression via the HIF1α/HES1/SIRT1 complex and prevented CHDs induced by pathological hypoxia. Our results indicate that spatial differences in oxygenation of the developing heart serve as signals to control CPC expansion and cardiac morphogenesis. We propose that physiological hypoxia coordinates homeostasis of CPCs, providing mechanistic explanations for some nongenetic causes of CHD.

Abnormal Notch-Hes Signaling Pathways and Acute Leukemia -Review

The abnormal activation of Notch signaling is closely related to the development of acute leukemia (AL). The core elements of the Notch signaling system include Notch receptors, Notch ligands, CSL DNA-binding proteins, and effectors like target genes. Any factors, which affect ligands, receptors, signal transducers and effectors, can influence the signal transduction of Notch signaling greatly. Based on the role of Notch signaling in AL, several targeted drugs against Notch upstream signaling have been developed. However, due to the complexity and pleiotropic effects of Notch upstream signaling, these targeted drugs display strong side effects. Thus, Hes (Hairy Enhancer of Split) factors as a primary Notch effector, also play an important role in the pathogenesis of AL. This review summarizes recent progresses on Notch-Hes signaling in AL, hopping to provide references for further excavation of the Notch-Hes signaling, and lay foundations for developing the next generation of targeted drugs.

P1.02-020 The Effect of EGF-Pathway Targeted Immunization (EGF PTI) on STAT3 and Cancer Stem Cells in EGFR Mutant NSCLC Cells

The vast majority of advanced non-small cell lung cancer (NSCLC) patients with EGFR mutant tumors will develop disease progression following successful treatment with an EGFR tyrosine kinase inhibitor (TKI). Resistance to EGFR-TKIs is due to various mechanisms, such as the secondary mutation (T790M) or the activation of alternative pathways (MET, AXL). What has not been fully appreciated is that EGFR blockade induces an imbalance in favor of survival, increases activity of STAT3 and enriches lung CSCs through Notch3-dependent signaling. EGF-PTI was designed to elicit an antibody response against EGF, in order to reduce EGF receptor signaling and limit tumor growth. We have explored whether EGF-PTI alone or in combination with EGFR TKIs may efficiently inhibit STAT3 and target CSCs. EGF PTI was provided by Bioven (Europe) Ltd. Gefitinib, erlotinib and the recently FDA-approved third-generation EGFR TKI, AZD9291 (osimertinib) were purchased from Selleck chemicals. Western blotting was used to assess the effect of the drugs on ERK, AKT and STAT3 phosphorylation and on Notch and PARP cleavage in EGFR (del19) mutant NSCLC PC9 cells and gefitinib-resistant PC9-GR4 cells. PC9-GR4 cells have been established in our lab and harbor the resistant T790M mutation (T790M+). The protein expression of AXL and CSCs markers such as HES1 (downstream effector of Notch) and Bmi1 was also examined. Gefitinib, erlotinib or AZD9291 suppressed EGFR, ERK1/2 and AKT phosphorylation in PC9 cells but increased STAT3 phosphorylation on the tyrosine residue 705 in both PC9 and PC9-GR4 cells. EGF-PTI suppressed STAT3, EGFR and ERK1/2 and the combination of each of the three EGFR TKIs with EGF-PTI lead to more potent inhibition of STAT3, EGFR and ERK1/2. The EGF-PTI induced AKT phosphorylation was reversed when EGF PTI was combined with EGFR TKIs. Interestingly, EGF-PTI blocked Notch cleavage and decreased the expression of HES1. The expression of Bmi1 and AXL were also attenuated with EGF PTI and apoptosis was enhanced through the induction of PARP cleavage. EGF PTI may reverse mechanisms of resistance to single EGFR inhibition and the combination of EGF PTI with EGFR TKIs efficiently inhibits downstream signaling pathways in T790M+ cells. Based on these results, the design of a proof-of-concept trial with the combination of EGF PTI with gefitinib for the first line treatment of EGFR mutant NSCLC patients is in progress.

Notch/Hes signaling and miR-9 engage in complex feedback interactions controlling neural progenitor cell proliferation and differentiation.

ABSTRACTCanonical Notch signaling has diverse functions during nervous system development and is critical for neural progenitor self-renewal, timing of differentiation and specification of various cell fates. A key feature of Notch-mediated self-renewal is its fluctuating activity within the neural progenitor cell population and the oscillatory expression pattern of the Notch effector Hes1 and its target genes. A negative feedback loop between Hes1 and neurogenic microRNA miR-9 was found to be part of this oscillatory clock. In a recent study we discovered that miR-9 expression is further modulated by direct binding of the Notch intracellular domain/RBPj transcriptional complex to the miR-9_2 promoter. In turn, miR-9 not only targets Hes1 but also Notch2 to attenuate Notch signaling and promote neuronal differentiation. Here, we discuss how the two interwoven feedback loops may provide an additional fail-save mechanism to control proliferation and differentiation within the neural progenitor cell population. Furthermore, we explore potential implications of miR-9-mediated regulation of Notch/Hes1 signaling with regard to neural progenitor homeostasis, patterning, timing of differentiation and tumor formation.

Role of Mir-34 Upregulation in Disruption of c-Myc, c-Myb and NOTCH Signaling in Diamond-Blackfan Anemia

Approximately 53% of patients with Diamond-Blackfan Anemia (DBA) have mutations in one of nine ribosomal protein components (RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPL5, RPL11 and RPL35A), with RPS19 being the most commonly affected (25% of all patients). Mutations found in patients result in protein haploinsufficiency and subsequent ribosomal stress, which increases the expression and activity of p53, leading to a failure of HSC differentiation during erythropoiesis. While RPS19-/- mice are not viable, we have generated a model system using different shRNA constructs to generate varying degrees of RPS19 knockdown in cultured cells and animal models. Cultured cells (including CD34+ HSCs derived from fetal liver or cord blood) recapitulate features of DBA erythropoietic failure in vitro and in vivo, including p53 upregulation and defects in erythrocyte differentiation, while anemias and skeletal defects are observed in zebrafish models.Using next-generation miRNA sequencing on the Illumina HiSeq2000 platform, we identified a number of miRNAs differentially expressed between human fetal liver CD34+ HSCs expressing normal or reduced RPS19. Preprocessing, alignment, and miRNA quantification were carried out using miRDeep2 [1] software. Differential expression analysis was performed with DESeq2 [2], which tests for differential expression based on a model using negative binomial distribution for count data from high-throughput sequencing assays. Candidates underwent a second round of screening based on robustness of expression differential (fold increase/decrease, variation of fold increase/decrease across replicates, and increase/decrease relative to RPS19 expression) and possible links to hematopoiesis and erythropoiesis, as reported in the scientific record. All three miR-34 isoforms (a, b and c) are robustly upregulated upon RPS19 reduction, with the extent of miR-34 upregulation being inversely proportional to RPS19 expression. When RPS19 protein expression is suppressed by approximately 50% (as observed in DBA patients), miR-34a, b and c isoforms are upregulated by 3.2, 2.2 and 2.6 fold respectively. In other systems, increased miR-34 results in modulation of a number of factors linked to erythropoiesis, including downregulation of c-Myc, Myb and NOTCH signaling. Genome-wide transcriptional analysis using next-generation sequencing on the Illumina HiSeq platform in CD34+ HSCs indicates c-Myc (4.2 fold), c-Myb (2.8 fold) and a number of NOTCH effectors [SIRT1 (1.8 fold) andHES1 (1.9 fold)] are indeed downregulated at the transcriptional level. Results were validated with qRT-PCR and western blot analysis, however, while transcriptional modulation fully accounts for c-Myc downregulation at the mRNA and protein level, we propose that c-Myb protein levels are modulated both transcriptionally and post-translationally, as protein expression is significantly further downregulated than mRNA message (6.3 fold). We propose that miR-34 upregulation in RPS19-depleted cells occurs through p53 and initiates genomic changes incompatible with erythropoiesis through downregulation of transcription factors c-Myc, c-Myb and the NOTCH signaling pathway. DisclosuresNo relevant conflicts of interest to declare.

Hes4: A potential prognostic biomarker for newly diagnosed patients with high-grade osteosarcoma.

Prognostic biomarkers for osteosarcoma (OS) at the time of diagnosis are lacking. Necrotic response of OS to preoperative chemotherapy correlates with survival and is determined 3-4 months after diagnosis. The purpose of this study is to identify biomarkers that will stratify patients into good or poor responders to chemotherapy at diagnosis and determine the role of potential biomarkers in OS pathogenesis. Because OS may be caused by disruptions of osteogenic differentiation, and the Notch pathway is one regulator of bone development, we examined the link between Notch effectors, OS differentiation, and OS outcome. We probed the R2:Genomics Analysis and Visualization Platform for RNA expression levels of Notch targets in mixed high-grade OS pretreatment biopsies. We used human OS cell lines in vitro and in mice to determine the role of the Notch target hairy/enhancer of split 4 (Hes4) in OS. We found that in OS patients, high expression of Hes4 is correlated with decreased metastasis-free and overall survival. Human OS cells that overexpress Hes4 are more immature and have an increased invasive capacity in vitro. This was not universal to all Notch effectors, as Hes1 overexpression induced opposing effects. When injected into NSG mice, Hes4-overexpressing OS cells produced significantly larger, more lytic tumors and significantly more metastases than did control cells. Hes4 overexpression promotes a more aggressive tumor phenotype by preventing osteoblastic differentiation of OS cells. Hes4 expression may allow for the stratification of patients into good or poor responders to chemotherapy at diagnosis.