Targeting Notch Signaling Research Articles

Abstract 2798: Identification of gamma secretase inhibitor (GSI)-responsive GBM patients: Involvement of NOTCH, p53, and PI3K/MAPK signaling

Abstract Glioblastoma (GBM) is the most common and lethal primary intracranial tumor. The recent standard-of-care treatment consisting of surgery, followed by radiotherapy and temozolomide, just prolongs the median survival period of GBM patients to 14.6 months. What impedes the survival is due to the extensive heterogeneity among GBM patients at both the cellular and the molecular levels. In our study, we focused on the precision medicine strategy targeting NOTCH signaling. According to recent Phase I/II clinical trial results, only a few GBM patients showed satisfactory response to GSIs. However, little is known regarding how to distinguish GSI-responsive patients. Here, we illustrated that it required (1) high NOTCH activity, (2) wild-type p53 and elevated P53 activity, (3) increased MAPK activity, and (4) decreased PI3K/AKT activity for GBM to respond to GSIs. In our study, we tested a series of patient-derived primary glioma stem cells (GSCs) to two GSIs (RO4929097 and BMS-708163). GSI-sensitive lines were identified and GSI compounds impaired cell viability and sphere formation ability in these cell lines. GSIs also induced cell death of the sensitive GSCs. To find out the signature of the GSI-sensitive GSCs, we did enrichment and correlation analyses against RNA sequencing, RPPA, and methylation data of the GSI-sensitive and resistant GBM lines. We found that GSI-sensitive GSCs harbored significantly elevated Notch activity (high NOTCH1 and NICD1 expression, and low Notch1 methylation). Interestingly, GSI-responsive cells also possessed increased P53 activity (increased expression of P53 targets-P21, BAX and TIGAR), high wild-type p53 frequencies, increased MAPK activity (elevated p-Erk1/2-Thr202/Tyr204 expression), and decreased PI3K/AKT activity (high PTEN expression and low p-AKT-Thr308, RAPTOR, p-4E-BP1-Ser65 expression) (P<0.05). In an attempt to strengthen GSI effect, we showed that MDM2 inhibitor Nutlin-3 (retards P53 degradation and increases P53 activity) combining RO4929097 exhibited synergistic effect on suppressing cell viability of GSI-sensitive cells with wild-type p53. In summary, we elucidated the genetic characteristics (high NOTCH, wild-type p53, high MAPK, and low PI3K/AKT) of GSI sensitive GSCs. Co-administration of Nutlin-3 enhanced GSI function in the subset of GBMs. Our findings indicated genetic markers could be used to identify patients responsive to GSIs, providing a new therapeutic strategy to treat this population of GBM patients. Citation Format: Chen Zhang, Martinez-Ledesma Juan, Jie Ding, Feng Gao, Shaofang Wu, Xiaolong Li, Erik Philip Sulman, Dimpy Koul, WK Alfred Yung. Identification of gamma secretase inhibitor (GSI)-responsive GBM patients: Involvement of NOTCH, p53, and PI3K/MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2798. doi:10.1158/1538-7445.AM2017-2798

RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells.

Endothelial-mesenchymal transition (EndMT) is an essential mechanism in the cardiovascular system, for both cardiovascular development and cardiovascular diseases (CVDs). Recent studies indicate that runt-related transcriprunt-related transcription factor 3 (RUNX3) contributes to EndMT and endothelial cell dysfunction. However, the underlying molecular mechanism remains unknown. The present study was designed to investigate the role of RUNX3 in EndMT and endothelial cell function, and to elucidate the underlying molecular mechanism. Human cardiac microvascular endothelial cells (HCMECs) were incubated in strictly controlled hypoxic conditions (1% O2). HCMECs were cultured under normoxic conditions (21% O2), and then moved to a strictly controlled hypoxic environment (1% O2). Under this hypoxic condition, the cells were transfected with the lentiviral vector containing RUNX3 or an empty lentiviral vector for 8 h. After the cells were cultured under hypoxic conditions for 4 days, CD31 and α-smooth muscle actin colocalization were assessed by immunofluorescence microscopy. Transwell migration and tube formation assays were used to examine the migration and angiogenesis ability. RT-qPCR and western blotting were used to determine the expression of molecules involved in EndMT. Hypoxia induced the transition of HCMECs to mesenchymal cells and markedly promoted tube formation and cell migration. Transforming growth factor-β (TGF-β) and Notch signaling were activated during the hypoxia-induced EndMT of HCMECs. RUNX3 knockdown attenuated EndMT of HCMECs, promoted angiogenic phenotype, and reduced endothelial cell migration. In conclusion, our results showed that RUNX3 knockdown attenuated hypoxia-induced EndMT and reversed endothelial cell functions. RUNX3 is a common downstream target of TGF-β and Notch signaling, and may be a novel therapeutic target for treating CVD mediated by EndMT.

The effect of blocking Notch signaling by γ-secretase inhibitor on allergic rhinitis

ObjectiveThis study aimed to investigate the effect of blocking Notch signaling by γ-secretase inhibitor (GSI) on allergic rhinitis. MethodGSI, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT) was administered to ovalbumin-induced AR mice models intranasally. We observed symptoms of sneezing and nose rubbing. To detect the inflammatory state, the serum OVA-specific-IgE, IFN-γ, IL-4, and IL-5 were analyzed by ELISA, and Th cell cytokines in nasal mucosa were analyzed by RT-PCR, including T-bet, IFN-γ, GATA-3, IL-4, and IL-5. In addition, hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) were applied for histopathological examination. As for the evaluation of Notch signaling, we analyzed the Notch-1, Notch signaling target Hes-1, and Hes-5 in mucosa by RT-PCR, besides, used western blotting and immunohistochemistry to assess NICD (Notch intracellular domain). ResultsThe results showed that the DAPT ameliorated the development of AR and suppressed Th2 cytokine levels significantly, alleviating eosinophils infiltration and goblet cells metaplasia, suggesting that the GSI can regulate Th2 response and weaken airway inflammation in AR. ConclusionOur findings provide evidence that blocking Notch signaling by GSI offers high value in treating AR.

Essential roles of Tbx3 in embryonic skin development during epidermal stratification.

Stepwise differentiation of epidermal cells is essential for development of stratified epithelium, but the underlying mechanisms remain unclear. Here, we show that Tbx3, a member of the T-box family of transcription factors, plays a pivotal role in this mechanism. Tbx3 is expressed in both basal and suprabasal cells in the interfollicular epidermis of mouse embryos. Epidermis-specific Tbx3 conditional knockout (cKO) embryos are small in size and display a thinner epidermis with an impaired barrier function. In the Tbx3 cKO epidermis, keratin 5-positive undifferentiated cells, which reside in both basal and suprabasal layers of wild-type embryos, are localized exclusively in the basal layer. In addition, mRNA expression levels of granular cell markers are increased in the Tbx3 cKO epidermis, suggesting that Tbx3 prevents premature differentiation of spinous cells. We further show that Tbx3 maintains the proliferative potential of basal cells and ensures their planar-oriented cell division. Moreover, Tbx3 is shown to be required for the expression of Hes1, a well-known Notch signaling target protein that is essential for epidermal development. We therefore propose that Tbx3 functions upstream of Hes1 to regulate proliferation and differentiation of basal and suprabasal cells during epidermal development.

MicroRNA-34a regulates liver regeneration and the development of liver cancer in rats by targeting Notch signaling pathway.

ObjectiveThis study aimed to investigate the role of microRNA-34a (miR-34a) in regulating liver regeneration (LR) and the development of liver cancer in rats by targeting Notch signaling pathway.MethodsThirty male Sprague-Dawley (SD) rats were randomly assigned into partial hepatectomy (PH) group and sham hepatectomy (SH) group. Hematoxylin and eosin (HE) staining was used to observe the histological change in liver tissues. Enzyme-linked immunosorbent assay (ELISA) was used to measure the serum tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) levels. Dual-luciferase reporter gene assay was performed to examine whether miR-34a targeted Notch1 gene. Human liver cancer Huh7 cells were transfected and divided into blank, negative control (NC), miR-34a mimics and miR-34a inhibitors groups. MTT and flow cytometry were used to detect cell growth, and cell cycle and apoptosis, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied detect to the expressions of miR-34a and Notch receptor mRNA. Western blotting was performed to detect the protein expressions of Notch receptors, P21, Bax, Bcl-2 and Bcl-xL. Tumor xenograft in nude mice was done to observe tumor formation in different groups.ResultsCompared to the SH group, miR-34a expression in liver tissues in the PH group decreased first and then increased to the normal level during LR. In early stage of LR, the expressions of Notch receptors and miR-34a were negatively correlated. Compared to the blank and NC groups, the cell growth was inhibited, cell cycle was mainly arrested in the G2/M phase and cell apoptosis rate increased in the miR-34a mimics group. Moreover, the expressions of miR-34a, P21 and Bax were up-regulated, while the expressions of Notch receptors, and Bcl-2 and Bcl-xL were down-regulated in this group. Additionally, the tumor growth in the miR-34a mimics group was reduced. The miR-34a inhibitors group showed contrary tendencies.ConclusionOur study demonstrates that miR-34a regulated LR and the development of liver cancer by inhibiting Notch signaling pathway.

Abstract A32: Upregulation of the Notch signaling pathway is associated with mortality and in vitro cell invasion in squamous cell carcinoma of the oral cavity

Abstract Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with low survival rates, which have not significantly improved despite advances in surgery and radiotherapy. Although human papillomavirus is a strong predictor of outcomes in oropharyngeal tumors, other HNSCC subtypes lack reliable prognostic and therapeutic biomarkers. The purpose of this study was to identify molecular targets to improve the management of oral cavity SCC (OSCC). We isolated RNA from 19 OSCCs treated surgically at UAB and evaluated gene expression using the NanoString nCounter system and PanCancer Pathways analysis module. Results were confirmed using data from the oral cavity anatomic subdivision of the Head and Neck Squamous Cell Carcinoma cancer study generated by the TCGA Research Network. Associated in vitro phenotype was assessed in HNSCC cell lines UM-SCC1, UM-SCC6 and FaDu treated with the activating Notch ligand DLL4 and included evaluation of gene expression, proliferation, cell cycle, migration, invasion, and radiosensitivity. We identified a significant association between upregulation of the Notch pathway and mortality in both the UAB and TCGA OSCC sample sets. In vitro activation of Notch signaling altered transcription in a pattern similar to that found in patient samples from the mortality cohorts. HNSCC cells with Notch activation had increased expression of pro-invasive genes FGF1, IL1B, SIX1, and CCNA1. In addition, Notch activated cells displayed increased cell migration and invasion. Proliferation, cell cycle distribution, and radiosensitivity were unchanged in ligand-treated cells. Our results identify a subset of OSCC patients who have increased Notch pathway activity and worsened outcomes. These clinical findings may reflect a more invasive cell phenotype driven by Notch-mediated changes in transcription. Additional studies are needed to fully elucidate the mechanism underlying this association and to investigate the efficacy of therapies targeting Notch signaling in OSCC. Citation Format: Alice N. Weaver, Tiffiny S. Cooper, Deborah Della Manna, Shi Wei, Eben L. Rosenthal, Eddy S. Yang. Upregulation of the Notch signaling pathway is associated with mortality and in vitro cell invasion in squamous cell carcinoma of the oral cavity. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A32.

Notch Signaling and Maintenance of SOX2 Expression in Rat Anterior Pituitary Cells.

After publication of reports describing the presence of stem/progenitor cells among non-hormone-producing cells in the pituitary, the mechanism responsible for proliferation and differentiation generated considerable interest. Several studies have suggested that Notch signaling is involved. In the present study, we examined the histochemical relationship between Notch signaling molecules and the transcription factor SOX2 in rat pituitary. Combined in situ hybridization and immunohistochemistry showed that Notch2 mRNA and SOX2 were co-expressed at embryonic day 14.5 in most cells in the adenohypophyseal primordium. In adult rat pituitary, double immunohistochemistry showed that SOX2 and either Notch2 or the Notch signaling target HES1 were co-localized within cells with large oval nuclei in both the marginal cell layer and cell aggregates in the main part of the anterior lobe, which are believed to be stem cell niches. Furthermore, when the Notch signaling inhibitor DAPT was added to a primary culture of adult rat anterior pituitary cells, the proportion of SOX2-expressing cells within Notch2-positive cells was approximately 30% lower. These findings suggest that Notch signaling has a role in maintaining the stemness of precursor cells in the adult rat pituitary gland.

In This Issue

HomeCirculation ResearchVol. 119, No. 12In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published9 Dec 2016https://doi.org/10.1161/RES.0000000000000130Circulation Research. 2016;119:1255Clonal Expansion of Plastic VSMCs in Disease (p 1313)Download figureDownload PowerPointVascular smooth muscle cells vary in their propensity for lesion formation, say Chappell et al.Vascular smooth muscle cells (VSMCs) in blood vessel walls are generally quiescent, but after a vessel injury, or during the formation of atherosclerotic lesions, these cells contribute to lesion formation by undergoing a phenotypic switch to become proliferative and migratory. However, it is unclear whether or not all VSMCs have equal capacity for lesion formation. To investigate this question, Chappell and colleagues expressed a multicolored reporter specifically in the VSMCs of atherosclerotic mice so that each cell was randomly labeled with one of four colors. The team showed that, in contrast to the multicolored vessel walls, atherosclerotic plaques contained regions that were largely monochromatic—suggesting that they were derived from single cells. These monochromatic plaque VSMCs also displayed a variety of phenotypes, including fibrous cap cells, core plaque cells, and macrophage-like cells. These results suggest that, during atherogenesis, individual VSMCs become highly proliferative and plastic. The team observed similar monochromatic lesions in mice whose vessels were physically injured (by ligation), though the phenotypic diversity of these VSMCs was less pronounced. The authors suggest that targeting the specific subset of highly proliferative VSMCs in vascular disease might curb lesions without adversely affecting healthy vessels.Notch Regulates Potassium Currents (p 1324)Download figureDownload PowerPointKhandekar et al discover that Notch signaling regulates potassium channel expression epigenetically.Cardiac arrhythmias can often arise from anomalies at junctions between the impulse-conducting Purkinje fibers and the cardiomyocytes. Aberrant activation of Notch signaling in the myocardium has been shown to convert cardiomyocytes into Purkinje-like cells and thus disrupt normal junction operations. Khandekar and colleagues, therefore, investigated how Notch signaling prompts this arrhythmia-inducing cell transition. They found that activation of Notch in left ventrical myocytes attenuated potassium currents in these cells, which in turn caused the cells’ action potentials to become prolonged—like those of Purkinje fibers. This potassium current suppression was caused by a reduction in the expression of potassium channel subunits, which itself was caused by removal of activating epigenetic marks from the promoters of the subunit genes. The team also showed that, in mice, an increase in Notch activity during heart failure repressed the expression of potassium channels by the same epigenetic means. Thus, targeting Notch signaling may be a potential therapeutic strategy for preventing arrhythmias caused by infarctions or other pathological conditions.Hypertension and Cerebral Perfusion (p e140)Download figureDownload PowerPointWarnert et al find evidence supporting the selfish brain hypothesis of hypertension.High blood pressure affects a quarter of the world’s population, but in 95% of these cases, the underlying causes remain unknown. It is clear that, in most patients, increased sympathetic nerve activity (SNA) drives the development of hypertension, but the factors that increase SNA have not been identified. One view is that SNA-induced hypertension is a safety mechanism that ensures sufficient blood flow to the brain. Support for this “selfish brain hypothesis” has come from the finding that, in contrast to other arteries, high resistance in the vertebral arteries of the neck is strongly correlated with hypertension. Moreover, clamping the vertebral arteries of rats increases the animals’ SNAs. Warnert and colleagues have now investigated the selfish brain hypothesis in humans. In their study, MRI scans showed that patients with vertebral artery hypoplasia (VAH) were more likely to have frank or borderline hypertension. Importantly, they also showed that, while patients with hypertension tended to have both increased vascular resistance and increased SNA, patients with borderline hypertension tended to have increased vascular resistance alone. This indicates that vascular resistance precedes the increase in SNA. If the selfish brain hypothesis is correct, as these results suggest, then aggressive blood pressure-lowering treatments require more careful consideration, say the authors. Previous Back to top Next FiguresReferencesRelatedDetails December 9, 2016Vol 119, Issue 12 Advertisement Article InformationMetrics © 2016 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000130 Originally publishedDecember 9, 2016 PDF download Advertisement

PlexinD1 Is a Novel Transcriptional Target and Effector of Notch Signaling in Cancer Cells

The secreted semaphorin Sema3E controls cell migration and invasiveness in cancer cells. Sema3E-receptor, PlexinD1, is frequently upregulated in melanoma, breast, colon, ovarian and prostate cancers; however, the mechanisms underlying PlexinD1 upregulation and the downstream events elicited in tumor cells are still unclear. Here we show that the canonical RBPjk-dependent Notch signaling cascade controls PlexinD1 expression in primary endothelial and cancer cells. Transcriptional activation was studied by quantitative PCR and promoter activity reporter assays. We found that Notch ligands and constitutively activated intracellular forms of Notch receptors upregulated PlexinD1 expression; conversely RNAi-based knock-down, or pharmacological inhibition of Notch signaling by gamma-secretase inhibitors, downregulated PlexinD1 levels. Notably, both Notch1 and Notch3 expression positively correlates with PlexinD1 levels in prostate cancer, as well as in other tumor types. In prostate cancer cells, Sema3E-PlexinD1 axis was previously reported to regulate migration; however, implicated mechanisms were not elucidated. Here we show that in these cells PlexinD1 activity induces the expression of the transcription factor Slug, downregulates E-cadherin levels and enhances cell migration. Moreover, our mechanistic data identify PlexinD1 as a pivotal mediator of this signaling axis downstream of Notch in prostate cancer cells. In fact, on one hand, PlexinD1 is required to mediate cell migration and E-cadherin regulation elicited by Notch. On the other hand, PlexinD1 upregulation is sufficient to induce prostate cancer cell migration and metastatic potential in mice, leading to functional rescue in the absence of Notch. In sum, our work identifies PlexinD1 as a novel transcriptional target induced by Notch signaling, and reveals its role promoting prostate cancer cell migration and downregulating E-cadherin levels in Slug-dependent manner. Collectively, these findings suggest that Notch-PlexinD1 signaling axis may be targeted to impair prostate cancer cell invasiveness and metastasis.

The blockage of Notch signalling promoted the generation of polymorphonuclear myeloid-derived suppressor cells with lower immunosuppression

Myeloid-derived suppressor cells (MDSCs) mostly consisting of polymorphonuclear (PMN)-MDSCs and mononuclear MDSCs have been considered to play critical roles in immunosuppression, angiogenesis, invasion and metastases of various tumours. However, it is still unclear the regulated mechanisms underlying the generation and immunosuppression of two major MDSC subsets. Here, we report Notch signalling was inhibited significantly in tumour-bearing mouse MDSCs, in which PMN-MDSCs were the major population. MDSCs without recombination signal binding protein-Jк (RBP-J), the critical transcription factor mediating signalling from all four mammalian Notch receptors, reduced their ability of inhibiting the proliferation and activation of allogenic T cells. RBP-J-deficient MDSCs could not down-regulate the expression of co-stimulation molecules on dendritic cells (DCs). The antigen presentation capacity of DCs co-cultured with RBP-J-deficient MDSCs was not impaired in contrast to controls. Moreover, we show the blockage of Notch signalling could improve the generation of PMN-MDSCs but inhibit the production of mononuclear MDSCs both in vitro and in vivo. Stat3 pathway was suppressed in MDSCs blocked Notch signalling and Stat3 activation by IL-6 could reverse the phenotype and immunosuppression of Notch signalling–deficient MDSCs. Therefore, targeting Notch signalling may be an effective therapeutic strategy in tumour therapy.

Downregulation of Notch Signaling Pathway as an Effective Chemosensitizer for Cancer Treatment.

Despite remarkable progress in cancer treatment, development of drug resistance is still a big burden to eliminate all tumor cells and a mean cause for tumor recurrence. Recent studies have been revealed the contribution of many signaling pathways in acquisition of resistance to chemotherapy. Because of its potential in maintaining the balance between cell proliferation and apoptosis, Notch signaling pathway has mean relevance to various aspects of cancer biology, from cancer stem cells to tumor immunity to multidrug resistance. Therefore, Notch signaling pathway is an attractive target for cancer therapy because targeting Notch signaling could overcome multi drug resistance (MDR). This article will provide a brief overview of the published evidences in support of Notch targeting in reverting multidrug resistance as a safer and novel approach for the improvement of tumor treatment.

Cardiomyocyte Lineage Specification in Adult Human Cardiac Precursor Cells Via Modulation of Enhancer-Associated Long Noncoding RNA Expression

The mechanisms controlling differentiation in adult cardiac precursor cells (CPCs) are still largely unknown. In this study, CPCs isolated from the human heart were found to produce predominantly smooth muscle cells but could be redirected to the cardiomyocyte fate by transient activation followed by inhibition of NOTCH signaling. NOTCH inhibition repressed MIR-143/145 expression, and blocked smooth muscle differentiation. Expression of the microRNAs is under control of CARMEN, a long noncoding RNA associated with an enhancer located in the MIR-143/145 locus and target of NOTCH signaling. The CARMEN/MIR-145/143 axis represents, therefore, a promising target to favor production of cardiomyocytes in cell replacement therapies.

Targeting Notch signaling as a novel therapy for retinoblastoma.

Retinoblastoma is the most common intraocular malignancy of childhood. Notch plays a key role in retinal cells from which retinoblastomas arise, and we therefore studied the role of Notch signaling in promoting retinoblastoma proliferation. Moderate or strong nuclear expression of Hes1 was found in 10 of 11 human retinoblastoma samples analyzed immunohistochemically, supporting a role for Notch in retinoblastoma growth. Notch pathway components were present in WERI Rb1 and Y79 retinoblastoma lines, with Jag2 and DLL4 more highly expressed than other ligands, and Notch1 and Notch2 more abundant than Notch3. The cleaved/active form of Notch1 was detectable in both lines. Inhibition of the pathway, achieved using a γ-secretase inhibitor (GSI) or by downregulating Jag2, DLL4 or CBF1 using short hairpin RNA, potently reduced growth, proliferation and clonogenicity in both lines. Upregulation of CXCR4 and CXCR7 and downregulation of PI3KC2β were identified by microarray upon Jag2 suppression. The functional importance of PI3KC2β was confirmed using shRNA. Synergy was found by combining GSI with Melphalan at their IC50. These findings indicate that Notch pathway is active in WERI Rb1 and Y79, and in most human retinoblastoma samples, and suggest that Notch antagonists may represent a new approach to more effectively treat retinoblastoma.

Abstract 886: Notch signaling activates B-cell specific enhancers to drive oncogene targets in B-cell lymphoma

Abstract Recurrent gain-of-function mutations in genes encoding Notch receptors are associated with poor clinical outcome in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but genome-wide functional targets of Notch signaling in B cells have not been characterized. We identified genome-wide binding sites for Notch transcription factor (TF) complexes, using antibodies specific for intracellular NOTCH1 and its binding partner RBPJ, in CLL and MCL lymph node biopsies, and lymphoma cell lines. We employed a gamma-secretase inhibitor washout strategy to rapidly modulate mutant NOTCH1 protein activation in complementary lymphoma cell line models of ligand-dependent and ligand-independent Notch signaling, and performed RNA-sequencing and genome-wide enhancer acetylation profiling in both the “notch on” and “notch off” states. These data identify a common set of functional Notch target genes in B-cell lymphoma that includes both canonical Notch targets as well as B-cell-specific target genes. The latter are frequently associated with adjacent B-cell-specific enhancers that show direct Notch TF binding and Notch-signaling-dependent histone acetylation. Notch-activated target genes include MYC and other TFs implicated in B-cell lymphoma, and are significantly enriched for mediators of targetable oncogenic lymphoma signaling pathways, (B-cell receptor, toll-like receptor, JAK-STAT, MAP kinase, and G-protein signaling pathways, all FDR q-value < 1e-3 in KEGG and / or Reactome analyses). Many B-cell Notch target genes show significantly increased expression in lymph nodes from patients with NOTCH1 mutant vs. wild-type CLL. Notch target genes are also up-regulated in lymph node- versus peripheral blood-derived CLL cells, consistent with immunohistochemical evidence for microenvironment-dependent Notch signaling activation in the CLL lymph node. Combined small-molecule inhibition of Notch and B-cell receptor signaling showed a synergistic anti-proliferative effect in Notch-dependent lymphoma cells. Our data reveal the functional regulome of Notch signaling in small B-cell lymphoma, and may have implications for the role of Notch signaling in normal B-cell development. These findings suggest novel strategies for rational combination therapy in CLL and MCL. Citation Format: Russell J. Ryan, Jelena Petrovic, Dylan M. Rausch, Winston Lee, Michael Kluk, Laura Donohue, Shawn Gillespie, Jon C. Aster, Warren S. Pear, Bradley E. Bernstein. Notch signaling activates B-cell specific enhancers to drive oncogene targets in B-cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 886.

Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival.

ABSTRACTEhrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival.

Weaponizing human EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) for 21st century cancer therapeutics.

De-regulated EFEMP1 gene expression in solid tumors has been widely reported with conflicting roles. We dissected EFEMP1 to identify domains responsible for its cell context-dependent dual functions, with the goal being to construct an EFEMP1-derived tumor-suppressor protein (ETSP) that lacked tumor-promoting function. Exon/intron boundaries of EFEMP1 were used as boundaries of functional modules in constructing EFEMP1 variants, with removal of various module(s), and/or mutating an amino acid residue to convert a weak integrin binding-site into a strong one. A series of in vitro assays on cancerous features, and subcutaneous and intracranial xenograft-formation assays, were carried out for effects from overexpression of wild-type and variant forms of EFEMP1 in two glioma subpopulations characterized as tumor mass-forming cells (TMCs) or stem-like tumor initiating cells (STICs), where EFEMP1 showed cellcontext- dependent dual functions. One of the EFEMP1 variants was identified as the sought-after ETSP, which had a stronger tumor-suppression function in TMCs by targeting EGFR and angiogenesis, and a new tumor-suppression function in STICs by targeting NOTCH signaling and MMP2-mediated cell invasion. Therefore, ETSP may form the basis for further important research to develop a novel cancer therapy to treat many types of cancer by its tumor suppressor effect in the extracellular matrix compartment.

Dual Mechanism of Action of Resveratrol in Notch Signaling Pathway Activation in Osteosarcoma

Purpose: To demonstrate the effect of resveratrol on Notch signaling in MG-63 and U2OS osteosarcoma cell lines. Methods: Cell Counting Kit 8 reagent was used to analyze cell proliferation while TRIzol® reagent was employed for the extraction of total RNA. High Capacity cDNA reverse transcription chain reaction kit was used to transcribe 2 μg RNA. Western blot analysis was performed to examine Hes1 and Hey1 expression. Results: The results revealed that resveratrol treatment exhibited dual mechanisms of action on the activation of Notch signaling in osteosarcoma cells. The osteosarcoma cell lines, MG-63 and U2OS, when exposed to 20 μM concentration of resveratrol for 48 h showed significant toxicity compared to untreated cells. However, 30 μM concentration of resveratrol induced higher toxicity which was lethal to cell growth. The results from RT qPCR and Western blot data revealed a concentration-dependent effect of resveratrol on the expression of Notch signaling genes including Hes1, Hes5, Hey1, Hey2 and HeyL in U2OS cells. Treatment of U2OS cells with 20 μM concentration of resveratrol for 48 h induced a marked increase in the expression of Hes1, Hes5, Hey1, Hey2 and HeyL mRNA compared to the untreated cells. However, at a concentration of 30 μM, resveratrol inhibited the activation of Notch signaling pathway. This was evident by a decrease in the expression of Hes1, Hes5, Hey1, Hey2 and HeyL, Notch signaling target genes. Conclusion: Resveratrol plays an important role in the activation of Notch signaling pathway and may be of therapeutic benefit in the treatment of osteosarcoma. Keywords: Osteosarcoma, Dual action mechanism, Notch signaling pathway, Toxicity, Cell growth inhibition

Abstract 409: Notch Signaling in Bone Marrow-derived FSP-1 + Cells Mediates a Phenotypic Change in Smooth Muscle Cells Leading to AVF Failure

Background: sociological and economic costs of failed arteriovenous fistulas (AVF) are well known but mechanisms of AVF losses are controversial. We have shown that smooth muscle cells (SMCs) from anastomosed artery compose of ~50% of neointima cells in AVF. Arterial anastomoses are also infiltrated by bone marrow (BM)-derived FSP-1 + cells, potentially linking FSP-1 + cells to SMC activation. However, whether Notch/RBP-JK signaling involves in BM-derived FSP-1 + cells activation has not been studied. We propose that Notch/RBP-JK activation in BM-derived FSP-1 + cells causes a phenotype-switch of SMCs, resulting in SMC translocation from the artery to the venous anastomosis, forming neointima. Methods: we created CKD and AVFs in wild type and Notch knock out mice. The role of Notch signaling in activation and function of BM-derived FSP-1 + cells was explored. Activated SMCs were characterized by a loss of differentiation markers plus a gain in proliferation (PCNA+). Using BM transplantation, we examined if FSP-1 + cells lacking RBP-JK in CKD mice would suppress SMC phenotype switch and neointima formation in AVFs. Results: BM-derived FSP-1 + inflammatory cells in AVFs from CKD mice were 45% greater vs. results in pair-fed, control mice. In the artery anastomosis, increased infiltration of FSP-1 + cells led to loss of SMC differentiation markers, SMMHC, SM-22 and SMA-α. Activation of Notch signaling (N1ICD + and RBP-JK + ) raised expression of cytokines (IL-1β, MCP-1) and growth factors (PDGF-BB, bFGF2 and TGF-β1) in FSP-1 + cells, The cytokines and growth factors caused a SMC phenotype switch (characterized by loss of SMC markers), resulting in SMC migration and proliferation. RBP-JK KO in BM-derived FSP-1 + cells significantly decreased the production of cytokines and growth factors, suppressed SMC activation of and neointima formation in AVFs. Conclusions: Notch signaling in BM-derived FSP-1 + cells can induce a phenotypic switch in SMCs stimulating neointima growth. Targeting Notch signaling in FSP-1 + cells could improve AFV function.

447. Notch Activation Enchances Vascular Lineage Commitment of Cardiac Stem Cells

Notch signaling pathway is an evolutionary conserved system that control heart development and could be important for cardiac tissue renewal and regeneration after injury by controlling the maintenance and commitment of a cardiac stem cell compartment. However the precise cell targets of Notch signaling in the mammalian damaged heart remain poorly defined. OBJECTIVE: To investigate the functional role of Notch signaling in the regulation of cardiac stem cells (CSCs) commitment in ischemic mice myocardium. METHODS AND RESULTS: Notch 1 receptor, their ligands and markers of cardiomyocyte, SMC, and endothelial cell lineages were examined in undamaged and infarcted C57BL/6 mice hearts by immunofluorescence staining. We have found that the majority of c-kit+ CSCs were nested in the interstitium between cardiomyocytes, and expressed Notch 1 receptor. Ligand Jagged 1 is presented on cardiomyocyte and cardiac fibroblasts, localized near c-kit+Notch1+ CSCs. Conversely, Delta-like4 ligand expressed on the endothelial and smooth muscle cells of coronary vessels. No signs of Notch1 signaling activation (such as nuclear NICD localization) were detected in undamaged mice hearts. Acute myocardial infarction was accompanied by the increase in the total amount of c-kit + Notch1 + CSCs and the appearance of nuclear NICD localization in a number of CSCs showing an activation of Notch signaling. Cultivation of CSC in vitro on dishes coated with Jagged 1 ligand released NICD and activated expression of Notch target genes (Hes, Hey). Activation of Notch signaling upregulated expression of endothelial cell transcription factors Gata 4 and Vezf1 in CSCs but γ-secretase inhibitor prevented Notch signaling activation and endothelial cell commitment of CSCs. CONCLUSIONS: These results revealed that Notch signaling activation promote CSCs commitment toward vascular cells. These findings suggest that modulation of Notch signaling can be promising therapeutic strategy for the treatment of myocardial ischemic damage.

T cell restricted Notch signaling contributes to pulmonary Th1 and Th2 polarization during Cryptococcus neoformans infection

Abstract Notch is a key signaling pathway regulating T cell development, activation and function. Notch signaling has been shown to affect the differentiation and functional responses of Th cells in several models of infectious disease and therapeutic targeting of Notch signaling has been proposed as treatments for autoimmune diseases. However, the contribution of Notch signaling to Th cell differentiation and responses during fungal infections is unknown. To elucidate the role of Notch signaling during fungal infections we infected mice expressing a dominant negative Notch inhibitor specifically within mature T cells with the fungal pathogen Cryptococcus neoformans. Inhibition of Notch resulted in impaired leukocyte recruitment, failure to control fungal burden in the lungs and failure to prevent dissemination to the CNS. Furthermore, we show that inhibition of Notch signaling impaired both protective Th1 and non-protective Th2 anti-cryptococcal immune responses. Leukocyte cultures and T cells from Notch deficient mice produced less IFN-γ, IL-5 and IL-13 than WT cells and had reduced expression of Th1 and Th2 associated transcription factors, Tbet and Gata3. Treatment with the Notch inhibitor DAPT did not impair cytokine production in WT cells indicating that Notch impacted the differentiation of these cells rather than biasing the magnitude of responses. Th17 responses were largely unaffected by Notch; although we also measured an increase in the frequency of Tregs in Notch deprived C. neoformans infected mice. Overall, these data suggest that Notch signaling controls the differentiation of Th1 and Th2 cells during C. neoformans infection and that targeting Notch may inadvertently render hosts more susceptible to fungal infections.