Notch Agonists Research Articles

Engineering synthetic agonists for targeted activation of Notch signaling.

Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although there are several classes of Notch inhibitors, the mechanical force requirement for Notch receptor activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonist (SNAG) proteins by tethering affinity-matured Notch ligands to antibodies or cytokines that internalize their targets. This bispecific format enables SNAGs to "pull" on mechanosensitive Notch receptors, triggering their activation in the presence of a desired biomarker. We successfully developed SNAGs targeting six independent surface markers, including the tumor antigens PDL1, CD19, and HER2, and the immunostimulatory receptor CD40. SNAGs targeting CD40 increase expansion of central memory γδ T cells from peripheral blood, highlighting their potential to improve the phenotype and yield of low-abundance T cell subsets. These insights have broad implications for the pharmacological activation of mechanoreceptors and will expand our ability to modulate Notch signaling in biotechnology.

Interference of FZD2 suppresses proliferation, vasculogenic mimicry and stemness in glioma cells via blocking the Notch/NF‑κB signaling pathway.

Frizzled family protein 2 (FZD2) is widely associated with tumor development and metastasis. The present study aimed to gain an insight into the role and regulatory mechanism of FZD2 in glioma. The expression level of FZD2 in normal astrocyte and glioma cells was determined by reverse transcription-quantitative PCR and western blotting, and cell transfection was conducted for FZD2 expression knockdown. Malignant behaviors including cell proliferation, migration and invasion, vasculogenic mimicry (VM) and cell stemness were determined using Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine (EdU) staining, colony formation, wound healing, Transwell, 3D culturing and sphere formation assays. The expression levels of proteins related to stemness, epithelial-mesenchymal transition (EMT) and Notch/NF-κB signaling were measured by western blotting. Then, the Notch agonist, Jagged-1 (JAG), was adopted for rescue experiments. The results demonstrated that FZD2 was highly expressed in glioma cells. Interference of FZD2 expression suppressed the proliferation of glioma cells, as evidenced by the reduced cell viability and the number of EdU+ cells and colonies. Meanwhile, the reduced sphere formation ability and decreased protein expression of Nanog, Sox2 and Oct4 following FZD2 knockdown confirmed that FZD2 repressed cell stemness in glioma. Additionally, FZD2 knockdown suppressed the migration, invasion, EMT and VM formation capabilities of glioma cells, and also blocked the Notch/NF-κB signaling pathway. Furthermore, activation of Notch by JAG treatment partially reversed the aforementioned FZD2 knockdown-mediated changes in glioma cell malignant behaviors. In conclusion, FZD2 may contribute to glioma progression through activating the Notch/NF-κB signaling pathway, providing a plausible therapeutic target for the treatment of glioma.

Tanshinol ameliorates imiquimod-induced psoriasis by inhibiting M1 macrophage polarization through suppression of the notch signaling pathway

BackgroundPsoriasis is a common immune-related chronic inflammatory skin disease, often accompanied by significant itching, and once diseased, the course of the disease lasts for most of the lifetime. Tanshinol (TAN) is an active ingredient of Salvia miltiorrhiza, which possesses pharmacological effects such as anti-inflammatory and antioxidant properties. However, the effects of TAN on psoriasis have not been widely reported. Therefore, the aim of this study was to investigate the therapeutic effects and mechanisms of TAN in psoriasis.MethodsAn imiquimod (IMQ)-induced psoriasis mouse model was constructed and treated with different doses of TAN to observe the changes in skin lesion phenotype, macrophage polarization, inflammation and Notch signaling pathway in mice. Further removal of macrophages or inhibition or activation of Notch signaling pathway was performed to examine the changes in skin lesion phenotype, macrophage polarization, inflammation and Notch signaling pathway in mice. In addition, in vitro experiments verified that TAN regulates RAW264.7 macrophage polarization and cytokine secretion through the Notch pathway.ResultsThe results showed that TAN alleviated IMQ-induced skin lesions and pathological phenotypes in psoriasis mice and inhibited Notch signaling pathway and M1-type macrophage polarization. Moreover, macrophage clearance and Notch signaling pathway activation inhibited the effect of TAN on psoriasis. Further in vitro experiments showed that Notch agonists reversed the effects of TAN on macrophage polarization and inflammatory cytokines.ConclusionsCollectively, these findings suggest that TAN may exert a therapeutic effect on psoriasis by inhibiting the Notch signaling pathway and thus M1-type macrophage polarization.

Regenerative failure of intrahepatic biliary cells in Alagille syndrome rescued by elevated Jagged/Notch/Sox9 signaling

Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.

Lenalidomide improves H2O2-induced PC12 cell injury by blocking the Notch signaling pathway.

Lenalidomide (LEN) has been reported to exert antitumor, anti-inflammatory, anti-angiogenic and immunomodulatory activities. However, the effects of LEN in spinal cord injury (SCI) are yet to be fully elucidated. The present study was conducted to identify whether LEN has a healing effect on SCI and to determine the underlying mechanism of action. The viability of H2O2-stimulated PC12 cells was detected using a Cell Counting Kit-8 assay. The viability of PC12 cells treated with LEN was examined with an MTT assay. The level of lactate dehydrogenase (LDH) was measured using an LDH assay kit, while the levels of the oxidative stress-related factors malondialdehyde, superoxide dismutase, glutathione peroxidase and catalase in PC12 cells were determined using commercial kits. Oxidative stress-related proteins were examined via western blotting. PC12 cell apoptosis was observed using a TUNEL assay, while apoptosis markers and Notch signaling-related proteins were examined via western blotting. The results of the present study demonstrated that H2O2 decreased PC12 cell viability in a dose-dependent manner. However, treatment with LEN significantly improved the viability of H2O2-stimulated PC12 cells and alleviated H2O2-induced cytotoxic injury. Additionally, LEN treatment inhibited the oxidative stress and apoptosis induced by H2O2 in PC12 cells. Notch signaling-related protein expression was downregulated after LEN treatment in H2O2-stimulated PC12 cells. In addition, a Notch agonist reversed the effects of LEN on H2O2-induced oxidative stress and PC12 cell apoptosis. In summary, it was demonstrated that LEN alleviated the H2O2-induced injury of PC12 cells by blocking the Notch signaling pathway, suggesting the value of applying LEN to the treatment of SCI.

Intrahepatic cholangiocyte regeneration from an Fgf-dependent extrahepatic progenitor niche in a zebrafishmodel of Alagille Syndrome.

Alagille Syndrome (ALGS) is a congenital disorder caused by mutations in the Notch ligand gene JAGGED1, leading to neonatal loss of intrahepatic duct (IHD) cells and cholestasis. Cholestasis can resolve in certain patients with ALGS, suggesting regeneration of IHD cells. However, the mechanisms driving IHD cell regeneration following Jagged loss remains unclear. Here, we show that cholestasis due to developmental loss of IHD cells can be consistently phenocopied in zebrafish with compound jagged1b and jagged2b mutations or knockdown. Leveraging the transience of jagged knockdown in juvenile zebrafish, we find that resumption of Jagged expression leads to robust regeneration of IHD cells through a Notch-dependent mechanism. Combining multiple lineage tracing strategies with whole-liver three-dimensional imaging, we demonstrate that the extrahepatic duct (EHD) is the primary source of multipotent progenitors that contribute to the regeneration, but not to the development, of IHD cells. Hepatocyte-to-IHD cell transdifferentiation is possible but rarely detected. Progenitors in the EHD proliferate and migrate into the liver with Notch signaling loss and differentiate into IHD cells if Notch signaling increases. Tissue-specific mosaic analysis with an inducible dominant-negative Fgf receptor suggests that Fgf signaling from the surrounding mesenchymal cells maintains this extrahepatic niche by directly preventing premature differentiation and allocation of EHD progenitors to the liver. Indeed, transcriptional profiling and functional analysis of adult mouse EHD organoids uncover their distinct differentiation and proliferative potential relative to IHD organoids. Our data show that IHD cells regenerate upon resumption of Jagged/Notch signaling, from multipotent progenitors originating from an Fgf-dependent extrahepatic stem cell niche. We posit that if Jagged/Notch signaling is augmented, through normal stochastic variation, gene therapy, or a Notch agonist, regeneration of IHD cells in patients with ALGS may be enhanced.

ILC2 Cells Promote Th2 Cell Differentiation in AECOPD Through Activated Notch-GATA3 Signaling Pathway.

This study is to investigate the capacity of type 2 innate lymphoid cells (ILC2s) in regulating the Th2 type adaptive immune response of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The study enrolled healthy people, stable chronic obstructive pulmonary disease (COPD) patients, and AECOPD patients. Flow cytometry was used to detect Th2 and ILC2 cells in the peripheral blood. In addition, ILC2s from the peripheral blood of AECOPD patients were stimulated with PBS, IL-33, Jagged1, DAPT, IL-33+Jagged1, IL-33+DAPT, and IL-33+Jagged-1+DAP in vitro. The levels of cytokines in the culture supernatant were detected by ELISA and the culture supernatant was used to culture CD4 + T cells. The mRNA and protein levels of Notch1, hes1, GATA3, RORα, and NF-κB of ILC2s were detected by real-time PCR and Western blot. The proportion of Th2 and ILC2s was significantly increased in the peripheral blood of AECOPD patients, alone with the increased Notch1, hes1, and GATA3 mRNA levels. In vitro results showed that the mRNA and protein levels of Notch1, hes1, GATA3 and NF-κB were significantly increased after stimulation with Notch agonist, meanwhile, the level of type 2 cytokines were increased in the supernatant of cells stimulated with Notch agonist, and significantly promoted differentiation of Th2 cells in vitro. Disruption of Notch pathway weakened GATA3 expression and cytokine production, and ultimately affected the differentiation of Th2 cells. In conclusion, our results suggest that ILC2s can promote Th2 cell differentiation in AECOPD via activated Notch-GATA3 signal pathway.

Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway.

Acute myeloid leukemia (AML) is a common type of hematological malignancy that can progress rapidly. AML has a poor prognosis and a high incidence of relapse due to therapeutic resistance. Azelaic acid (AZA), a small molecular compound is known to exhibit antitumor effect on various tumor cells. This study aimed to evaluate the antiproliferative and immunoregulatory effects of AZA against AMLviathe activation of the notch signaling pathway. We found that AZA can inhibit the proliferation of AML cells. In addition, laser confocal microscopy showed AZA-treated AML cells began to swelling and undergo cytoplasmic vacuolization. Importantly, AZA promoted the proliferation of NK and T cells and increased the secretion of TNF-αand IFN-γ. AZA also increased the expression levels of CD107a and TRAIL in NK cells, and CD25 and CD69 in T cells to influence their activation and cytotoxic ability. AZA-treated NK cells can kill AML cells more efficiently at the single-cell level as observed under the microfluidic chips. Further mechanistic analysis using protein mass spectrometry analysis and Notch signaling reporter assay demonstrated that Notch1and Notch2 were up-regulated and the Notch signaling pathway was activated. Moreover, combining AZA with the Notch inhibitor, RO4929097, decreased the expression of Notch1and Notch2, and downstream HES1 and HEY1, which rendered AML cells insensitive to AZA-induced apoptosis and alleviated AZA-mediated cytotoxicity in AML. In vivo, AZA relieved the leukemic spleen infiltration and extended the survival. The percentage of CD3-CD56+NK cells and CD4+CD8+T cells as well as the secretion of cytotoxic cytokines was increased after the treatment of AZA. The overall findings reveal that AZA is a potential Notch agonist against AML in activating the Notch signaling pathway.

Attenuation of EGFL7 Expression Inhibits Growth Hormone-Producing Pituitary Adenomas Growth and Invasion.

Invasiveness of growth hormone-producing pituitary adenomas (GHPAs) causes difficulties in safe and complete adenoma removal during surgery and often leads to high recurrence. Epidermal growth factor-like domain 7 (EGFL7) has been shown to be able to promote tumor angiogenesis, growth, invasiveness, and metastasis through the Notch signaling pathway. It was previously demonstrated that EGFL7 was overexpressed in GHPAs. This study reports that EGFL7 and Notch2 (positive correlation with EGFL7) are overexpressed in invasive GHPA. A long-rank test (Kaplan-Meier method) shows that invasive GHPAs with EGFL7 strong expression results in reduced recurrence-free survival. Multivariate Cox regression analysis reveals that weak EGFL7 expression is an independent prognostic factor for recurrence-free survival. In addition, knockdown of EGFL7 expression suppresses proliferation and invasion of GH3 and GT1-1 cells in vitro. Moreover, attenuation of EGFL7 inhibits human GHPA growth in vivo. The data suggest that as a Notch agonist, EGFL7 may potentially be an appropriate novel molecular target for future development of GHPA medical therapy.

Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice

Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.

MiR424-5p functions as an anti-oncogene in cervical cancer cell growth by targeting KDM5B via the Notch signaling pathway

AimsAberrant expression of miRNAs exert the critical roles in carcinogenesis, including cervical cancer. Recent study corroborated the down-regulation of miR424-5p in uterine cervix adenocarcinoma. This research aimed to investigate the function and underlying mechanisms of miR424-5p in cervical cancer cell growth. Main methodsTissues samples were collected from patients with cervical cancer and healthy control. The expression levels of miR424-5p were determined by qRT-PCR. After transfection with miR424-5p mimics or inhibitor, cervical cancer cell proliferation and apoptosis were evaluated by WST-1 and flow cytometry assay, respectively. The underlying mechanism involved in aforementioned processes was also explored. Key findingsExpression of miR424-5p was notably decreased in cervical cancer tissues and cells. Overexpression of miR424-5p restrained cell proliferation and promoted cell apoptosis, but with little function in miR424-5p inhibitor-treated groups. Furthermore, KDM5B was identified as a direct target of miR424-5p as the evidence that miR-424-5p inhibited KDM5B expression and luciferase activity of KDM5B 3′-UTR. Here, KDM5B elevation majorly reversed miR424-5p-triggered inhibition in cell proliferation and increase in cell apoptosis. Moreover, silencing KDM5B expression also restrained cell growth. Additionally, miR424-5p overexpression inhibited the expression of Notch1 and Notch2, which was obviously rescued after KDM5B up-regulation. Simultaneously, blocking KDM5B also attenuated the activation of Notch pathway. Importantly, treatment with Notch agonist Jagged1 antagonized miR424-5p-mediated suppression on cell growth. SignificanceThis research suggests that miR424-5p may act as a novel anti-oncogene in cervical cancer by blocking cell growth through targeting KDM5B-Notch pathway. Accordingly, our study will support a promising therapeutic strategy against cervical carcinoma.

Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia.

Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML.

The challenge of targeting notch in hematologic malignancies.

Notch signaling can play oncogenic and tumor suppressor roles depending on cell type. Hematologic malignancies encompass a wide range of transformed cells, and consequently the roles of Notch are diverse in these diseases. For example Notch is a potent T-cell oncogene, with >50% of T-cell acute lymphoblastic leukemia (T-ALL) cases carry activating mutations in the Notch1 receptor. Targeting Notch signaling in T-ALL with gamma-secretase inhibitors, which prevent Notch receptor activation, has shown pre-clinical activity, and is under evaluation clinically. In contrast, Notch signaling inhibits acute myeloblastic leukemia growth and survival, and although targeting Notch signaling in AML with Notch activators appears to have pre-clinical activity, no Notch agonists are clinically available at this time. As such, despite accumulating evidence about the biology of Notch signaling in different hematologic cancers, which provide compelling clinical promise, we are only beginning to target this pathway clinically, either on or off. In this review, we will summarize the evidence for oncogenic and tumor suppressor roles of Notch in a wide range of leukemias and lymphomas, and describe therapeutic opportunities for now and the future.

Notch modulates human NK cell differentiation through regulation of inhibitory killer immunoglobulin-like receptor (KIR) expression and acquisition of effector function (TUM2P.890)

Abstract The Notch signaling pathway plays a substantial role on human NK cell development, however the role of Notch on KIR upregulation and acquisition of effector function has not been explored. To evaluate how Notch influences terminal differentiation, cord blood derived NK cells or sorted KIR- peripheral blood NK cells were cultured with IL-15 for 7 days in the presence or absence of gamma-secretase inhibitor, known to inhibit Notch signaling. Inhibition of Notch signaling significantly decreased KIR expression. Conversely, co-culturing the same cells with OP9 cells bearing Notch ligands enhanced KIR expression. Overexpression of the active portion of Notch on cord blood derived NK cells after 28 days of culture resulted in a 2-fold increase in KIR expression indicating that Notch signaling plays a direct, cell intrinsic, role in KIR regulation. By knocking down delta-like 1 (DLL1) on NK cells and co-culturing them with OP9 cells expressing Notch ligands we show that DLL1 mediated cis-inhibition controls KIR expression. Notch signaling also enhances CD16 upregulation that precedes KIR expression. More important than receptor expression is function. We found that Notch signaling induces increased cytolytic effector capacity and cytokine secretion, even on post-transplant samples where NK cell function is inherently defective. Given these attributes of Notch signaling, we propose Notch agonists can enhance NK cell maturation and tumor killing in a post-transplant setting.

Induction of Myogenic Differentiation of Human Mesenchymal Stem Cells Cultured on Notch Agonist (Jagged-1) Modified Biodegradable Scaffold Surface

Engineered scaffold surface provides stem cells with vital cues that could determine the eventual fate of stem cells. In this work, biodegradable poly(L-lactide-co-ε-caprolactone) (PLCL) scaffold conjugated with Notch agonist-Jagged-1(JAG) peptide (2.1 kDa) was prepared to initiate myogenic differentiation of human mesenchymal stem cells (hMSCs). The scaffold surface was activated with oxygen plasma and acrylic acid was engrafted via UV polymerization to form a surface bearing carboxylic groups. JAG peptide was subsequently immobilized onto the carboxylated scaffold surface. Surface chemistry and topography were examined using attenuated total reflection Fourier transform infrared, X-ray photoelectron spectroscopy, and atomic force microscopy. Quantitative real time polymerase chain reaction analysis revealed activation of the Notch pathway; furthermore, several specific markers associated with myogenic but not osteogenic differentiation were shown to be up-regulated in hMSCs cultured on the engineered surface. The pro-myocardial effect of surface bound JAG peptide was further affirmed via immunodetection of the distinct myocardial marker, cardiac troponin T. Collectively, our results suggest that PLCL conjugated JAG peptide is a viable strategy to enhance the functional potential of scaffolds to be used as a bioengineered cardiac patch in myocardial infarction repair.

The 4 Notch receptors play distinct and antagonistic roles in the proliferation and hepatocytic differentiation of liver progenitors

The Notch signaling pathway is involved in liver development and regeneration. Here, we investigate the role of the 4 mammalian Notch paralogs in the regulation of hepatoblast proliferation and hepatocytic differentiation. Our model is based on bipotential mouse embryonic liver (BMEL) progenitors that can differentiate into hepatocytes or cholangiocytes in vitro and in vivo. BMEL cells were subjected to Notch antagonists or agonists. Blocking Notch activation with a γ-secretase inhibitor, at 50 μM for 48 h, reduced cell growth by 50%. S-phase entry was impaired, but no apoptosis was induced. A systematic paralog-specific strategy was set using lentiviral transduction with constitutively active forms of each Notch receptor along with inhibition of endogenous Notch signaling. This assay demonstrates that proliferation of BMEL cells requires Notch2 and Notch4 activity, resulting in significant down-regulation of p27(Kip1) and p57(Kip2) cyclin-dependent kinase inhibitors. Conversely, Notch3-expressing cells proliferate less and express 3-fold higher levels of p57(Kip2). The Notch3 cells present a hepatocyte-like morphology, enhanced multinucleation, and a ploidy shift. Moreover, Notch3 activity is conducive to hepatocytic differentiation in vitro, while its paralogs impede this fate. Our study provides the first evidence of a functional diversity among the mammalian Notch homologues in the proliferation and hepatocytic-lineage commitment of liver progenitors.

Notch activation inhibits AML growth and survival: a potential therapeutic approach

Although aberrant Notch activation contributes to leukemogenesis in T cells, its role in acute myelogenous leukemia (AML) remains unclear. Here, we report that human AML samples have robust expression of Notch receptors; however, Notch receptor activation and expression of downstream Notch targets are remarkably low, suggesting that Notch is present but not constitutively activated in human AML. The functional role of these Notch receptors in AML is not known. Induced activation through any of the Notch receptors (Notch1-4), or through the Notch target Hairy/Enhancer of Split 1 (HES1), consistently leads to AML growth arrest and caspase-dependent apoptosis, which are associated with B cell lymphoma 2 (BCL2) loss and enhanced p53/p21 expression. These effects were dependent on the HES1 repressor domain and were rescued through reexpression of BCL2. Importantly, activated Notch1, Notch2, and HES1 all led to inhibited AML growth in vivo, and Notch inhibition via dnMAML enhanced proliferation in vivo, thus revealing the physiological inhibition of AML growth in vivo in response to Notch signaling. As a novel therapeutic approach, we used a Notch agonist peptide that led to significant apoptosis in AML patient samples. In conclusion, we report consistent Notch-mediated growth arrest and apoptosis in human AML, and propose the development of Notch agonists as a potential therapeutic approach in AML.

Notch/HES1 Implicated As a Tumor Suppressor Mechanism in Human AML in Vivo

Abstract 3524 Background:Although Notch signaling contributes to T cell leukemogenesis, the role of Notch in human AML is unclear. We and others have found that activation of Notch signaling inhibits AML growth and survival, e.g. a tumor suppressor like effect. However it is not known what the consequences of activating or inhibiting Notch signaling are in human AML in vivo. Approach:To determine whether Notch signaling would have growth inhibiting effects in vivo, we stably-transduced ML1 human AML cells with the constitutively-active forms of Notch1 and Notch2 (ICN1, ICN2), the common Notch target gene Hairy/Enhancer of Split 1 (HES1) or the pan-Notch inhibitor dominant-negative Mastermind-like (dnMAML) and performed in vivo competitive proliferation assays. Briefly, following transduction, each vector type were sorted to 50% GFP+ (containing the gene of interest) and GFP- (untransduced control cells). Groups of NSG mice were injected with these 50:50 mixtures of ML1 cells, and peripheral blood levels of GFP- and GFP+ cells were measured with flow cytometry for anti-human CD45 and GFP. Results:Peripheral blood engraftment of human CD45+ ML1 cells by week 5 was similar (2–5%) for ICN1, ICN2 and HES1 injected mice, but was significantly higher (23%) in dnMAML injected mice (Panel A). Similar to in vitro competitive proliferation assays, ICN1, ICN2 and HES1 all led to decreased relative numbers of GFP%+ cells, with 1%, 4% and 16% respectively (Panel B). Importantly, dnMAML had little effect on AML proliferation in vitro, however led to dramatic increases in GFP% as well as early morbidity and mortality due to increased leukemia burden, with 93% GFP+ (Panel B), demonstrating a selective advantage for dnMAML-expressing ML1 in vivo. When GFP+ (transduced) ML1 peripheral engraftment was directly compared to GFP- (parental CD45+) engraftment, the GFP- control cells had similar engraftment rates (2–5%) across groups of mice, while the GFP+ engraftment rates were significantly lower in ICN1, ICN2, and HES1 groups, but significantly higher in the dnMAML group (Panel C), demonstrating enhanced engraftment/proliferation in dnMAML-expressing cells. Conclusions:This suggests a previously unreported concept, namely that endogenous Notch ligands can inhibit human AML growth in vivo. This data supports the hypothesis that Notch behaves as a tumor suppressor in AML, and suggests the potential use of Notch agonists in human AML. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

In Vivo Blockade of Individual Notch Ligands and Receptors Provides a New Targeted Therapeutic Approach In Graft-Versus-Host Disease

Abstract 819Notch signaling is a cell-cell communication pathway with multiple functions in health and disease. Notch ligands of the Delta-like (Dll1, 3, 4) or Jagged (Jagged1, 2) family interact with one of four mammalian Notch receptors (Notch1-4), leading to proteolytic activation of the receptors by gamma-secretase. We have discovered a critical role for Notch signaling in the differentiation of pathogenic host-reactive T cells during graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT). Expression of the pan-Notch inhibitor DNMAML in donor T cells led to markedly reduced GVHD severity, without causing global immunosuppression (Blood 2011, 117(1): 299–308). These findings identify Notch signaling in alloreactive T cells as an attractive therapeutic target after allo-BMT. To explore preclinical strategies of Notch blockade in GVHD, we first assessed the effects of systemic pan-Notch inhibition with gamma-secretase inhibitors. In the B6 anti-BALB/c MHC-mismatched model of allo-BMT, administration of the gamma-secretase inhibitor dibenzazepine was as efficient as genetic strategies at blocking Notch target gene expression and production of inflammatory cytokines in donor T cells (IFN-γ, TNF-α, IL-2). However, dibenzazepine induced severe gastrointestinal toxicity after total body irradiation due to inhibition of both Notch1 and Notch2 in the gut epithelium. To avoid these side effects, we hypothesized that targeting individual Notch receptors or ligands could provide safe therapeutic Notch blockade after allo-BMT. Among the four mammalian Notch receptors (Notch1-4), donor alloreactive T cells expressed Notch1 and Notch2. Host dendritic cells expressed Notch ligands of the Jagged and Delta-like (Dll) families, with markedly increased Dll4 but not Jagged1/2 transcripts after total body irradiation. This suggested that blockade of Notch1 and/or Notch2 in T cells or Delta-like Notch ligands in dendritic cells could abrogate GVHD. To explore this possibility, we used specific monoclonal antibodies to neutralize Notch receptors and ligands in vivo after allo-BMT (Nature 2006, 444(7122):1083–7; Nature 2010, 464(7291): 1052–7). Combined blockade of Notch1 and Notch2 in vivo reduced the production of key inflammatory cytokines by alloreactive CD4+ and CD8+ T cells to a similar extent as DNMAML-mediated pan-Notch inhibition. Inhibition of Notch1 alone led to a large decrease in cytokine secretion, indicating that Notch1 is a dominant non-redundant Notch receptor in alloreactive T cells. Consistently, transplantation of Notch1-deficient but not Notch2-deficient B6 T cells allowed for decreased GVHD and improved survival in BALB/c recipients, similarly to global Notch inhibition by DNMAML. We then studied the consequences of inhibiting Dll1, Dll4 or both Dll1/Dll4 Notch ligands during acute GVHD. Combined Dll1/Dll4 blockade was as potent as DNMAML expression in decreasing cytokine production by alloreactive T cells, demonstrating that Delta-like and not Jagged ligands are the key Notch agonists at the alloimmune synapse. Dll4 inhibition was superior to Dll1 blockade in reducing cytokine production, abrogating GVHD, and prolonging recipient survival. Importantly, combined Dll1/Dll4 inhibition provided long-term protection against GVHD morbidity and mortality, while avoiding severe gastrointestinal side effects from Notch inhibition. Protection was observed even upon transient Dll1/Dll4 blockade during 1–2 weeks after transplantation. Altogether, our data suggest that Notch1 and Dll4 preferentially interact during alloreactive T cell priming and identify novel strategies to safely and efficiently target individual elements of the Notch pathway after allo-BMT. Humanized antibodies against Notch receptors and ligands were designed to block both mouse and human proteins, thus our preclinical work could lead to new strategies for GVHD control in human patients. Disclosures:Shelton:Genentech Inc.: Employment. Yan:Genentech Inc.: Employment. Siebel:Genentech Inc.: Employment.

Notch/HES1-mediated PARP1 activation: a cell type–specific mechanism for tumor suppression

AbstractNotch signaling plays both oncogenic and tumor suppressor roles, depending on cell type. In contrast to T-cell acute lymphoblastic leukemia (ALL), where Notch activation promotes leukemogenesis, induction of Notch signaling in B-cell ALL (B-ALL) leads to growth arrest and apoptosis. The Notch target Hairy/Enhancer of Split1 (HES1) is sufficient to reproduce this tumor suppressor phenotype in B-ALL; however, the mechanism is not yet known. We report that HES1 regulates proapoptotic signals by the novel interacting protein Poly ADP-Ribose Polymerase1 (PARP1) in a cell type–specific manner. Interaction of HES1 with PARP1 inhibits HES1 function, induces PARP1 activation, and results in PARP1 cleavage in B-ALL. HES1-induced PARP1 activation leads to self-ADP ribosylation of PARP1, consumption of nicotinamide adenine dinucleotide+, diminished adenosine triphosphate levels, and translocation of apoptosis-inducing factor from mitochondria to the nucleus, resulting in apoptosis in B-ALL but not T-cell ALL. Importantly, induction of Notch signaling by the Notch agonist peptide Delta/Serrate/Lag-2 can reproduce these events and leads to B-ALL apoptosis. The novel interaction of HES1 and PARP1 in B-ALL modulates the function of the HES1 transcriptional complex and signals through PARP1 to induce apoptosis. This mechanism shows a cell type–specific proapoptotic pathway that may lead to Notch agonist–based cancer therapeutics.