scholarly journals The Role of Notch in Vascular Endothelial Cell-Mediated Protection of AML Precursors from Targeted Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2750-2750
Author(s):  
Quy Le ◽  
Soheil Meshinchi ◽  
Brandon Kenneth Hadland ◽  
Roland B. Walter ◽  
Irwin D. Bernstein
Keyword(s):  
Cell Viability ◽  
Notch Signaling ◽  
Liquid Culture ◽  
Treatment Effectiveness ◽  
Conflicts Of Interest ◽  
Initial Period ◽  
Vascular Endothelial Cell ◽  
Significant Risk ◽  
Transcriptional Analysis ◽  
Notch 1

Abstract Despite advances in the treatment of acute myeloid leukemia (AML), disease relapse following an initial period of remission remains a significant risk, suggesting the existence of resistant leukemic precursors. Recent studies indicate resistance may be imparted to the leukemic precursors by the hematopoietic niche within the bone marrow. Previously, we have shown that a key component of the niche, endothelial cells (EC), can support long-term growth of AML precursors in culture. Here, we show that Notch is a novel target to disrupt the protective AML-EC interactions and enhance treatment effectiveness. In preliminary studies of AML with FLT3 mutations, we demonstrated that leukemic precursors from primary human AML patient samples are protected from treatment with a small molecule FLT3 inhibitor, AC220, when cultured in the presence of EC. We assayed cell viability of AML cells following 3 days of treatment with AC220 (100nM) or control (DMSO) in EC co-culture or liquid culture, and found that cells survived AC220 treatment better in EC co-culture compared to liquid culture (average percent cell viability from 3 primary FLT3-ITD+ AML patient samples relative to DMSO control: liquid culture (3 ± 3%) vs. EC co-culture (35 ± 19%), p=0.04). To identify genes involved in conferring protection to AML cells, we performed genome-wide transcriptional analysis of AML cells following 2 days of treatment with AC220 or DMSO in EC co-culture. EdgeR was used to assess differences in gene enrichment across cell populations. We found 1171 and 555 genes were increased and decreased, respectively, in AC220-treated population compared to DMSO control-treated population AML based on a 2-fold change and FDR<0.01. Amongst the differentially expressed genes, we found an enrichment of genes involved in Notch signaling pathway, including HES1, CDKN1A, and CCND1, in AML cells that survived treatment with AC220 compared to control. To evaluate a role for Notch signaling in EC-mediated growth of AML cells, we incubated AML cells in EC co-culture with inhibitory antibodies against Notch 1 and 2 receptors or control antibody for 2 weeks, and assayed the cells for colony-forming cell (CFC) activity. We found that inhibition of Notch signaling led to an increase in CFC formation. In contrast, when we combined AC220 with Notch 1 and 2 blocking antibodies in AML/EC co-culture, we found that inhibition of Notch signaling significantly reduced the number of total CFC and FLT3-ITD+ CFC compared to AC220 treatment combined with control antibody (p<0.005). These results suggest a role for Notch in EC-mediated protection of resistant AML precursors and thus offer a potential therapeutic strategy for sensitizing resistant precursors to targeted therapies. Disclosures No relevant conflicts of interest to declare.

Andrographolide Inhibits Proliferation of Colon Cancer SW-480 Cells via Downregulating Notch Signaling Pathway.

2020 ◽  
Vol 20 ◽  
Author(s):  
Imran Khan ◽  
Sadaf Mahfooz ◽  
Mohd Saeed ◽  
Irfan Ahmad ◽  
Irfan A. Ansari
Keyword(s):  
Cell Cycle ◽  
Colon Cancer ◽  
Notch Signaling ◽  
Signaling Pathway ◽  
Mtt Assay ◽  
Annexin V ◽  
Notch Signaling Pathway ◽  
Phase Cell ◽  
Ros Generation ◽  
Notch 1

Background: Recently Notch signaling pathway has gained attention as a potential therapeutic target for chemotherapeutic intervention. However, the efficacy of previously known Notch inhibitors in colon cancer is still unclear. The purpose of this study was to investigate the effect of andrographolide on aberrantly activated Notch signaling in SW-480 cells in vitro. Methods: The cytostatic potential of andrographolide on SW-480 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, morphology assessment and colony formation assay. The apoptotic activity was evaluated by FITC Annexin V assay, 4′,6-diamidino-2-phenylindole (DAPI), Hoechst, Rhodamine 123 and Mito Tracker CMXRos staining. Scratch assay for migratory potential assessment. 7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA) staining was used to evaluate the Reactive Oxygen Species (ROS) generation. Relative mRNA expression of Bax, Bcl2, NOTCH 1 and JAGGED 1 was estimated by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Cell cycle phase distribution was evaluated Annexin V-FITC/PI staining. Results: MTT assay demonstrated dose and time dependent cytoxicity of andrographolide on SW-480 cells. It also inhibited the migratory and colony forming potential of SW-480 cells. Furthermore, andrographolide also showed disruption of mitochondrial membrane potential and induced apoptosis through nuclear condensation. Flow cytometric evaluation showed andrographolide enhanced early and late apoptotic cells and induced upregulation of proapoptotic (Bax and Bad) and downregulation of antiapoptotic Bcl2 in treated SW-480 cells. Andrographolide augmented intracellular ROS generation and induced G0/G1 phase cell cycle arrest in colon cancer SW480 cells. Furthermore, andrographolide repressed the Notch signaling by decreasing the expression of NOTCH 1 and JAGGED 1. Conclusion: Our findings suggested that andrographolide constraint the growth of SW-480 cells through the inhibition of Notch signaling pathway.

scholarly journals POS0042 NOTCH 1 INHIBITION INCREASES OSTEOCLAST PROGENITOR ACTIVITY IN THE MOUSE MODEL OF RHEUMATOID ARTHRITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 226.3-226
Author(s):  
M. Filipović ◽  
A. Šućur ◽  
D. Flegar ◽  
Z. Jajić ◽  
M. Ikić Matijašević ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Notch Signaling ◽  
Neutralizing Antibodies ◽  
Osteoclast Differentiation ◽  
Notch Receptor ◽  
Notch 1 ◽  
Collagen Induced Arthritis ◽  
Notch Receptors

Background:Osteoclasts mediate periarticular and systemic bone loss in rheumatoid arthritis (RA). Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45+Ly6G-CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ cells, are specifically increased in arthritis. We previously identified an increased frequency of OCPs expressing Notch receptors in arthritic mice.Objectives:Several studies suggested that Notch signaling modulation affects the course of experimental arthritis. We aimed to determine the effects of Notch receptor signaling inhibition on OCP activity and arthritis severity in murine collagen-induced arthritis (CIA).Methods:Male C57/Bl6 and DBA mice were immunized with chicken type II collagen and treated with i.p. injections of anti-Notch 1 neutralizing antibodies (1mg/kg). Notch receptor 1 through 4 expression on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). Gene expression of Notch receptors, ligands and transcription targets as well as osteoclast differentiation genes RANK, cFos and cFms was determined by qPCR from tissues and sorted OCPs. FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL), in control, IgG, Jagged (Jag)1 or Delta-like (DLL)1 coated wells, with or without anti-Notch 1 antibodies. Research was approved by the Ethics Committee.Results:We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 25% and 40% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Seeding OCPs on DLL1 coated wells significantly increased while seeding on Jag1 coated wells significantly decreased osteoclastogenesis as reflected on the number of TRAP+ osteoclasts and expression of osteoclast differentiation genes. The addition of anti-Notch 1 antibodies to ligand-stimulated OCPs resulted in an increased number of TRAP+ osteoclasts, partially reversing Jag1 inhibition. In vivo treatment with anti-Notch 1 antibodies did not affect total OCP frequency, but increased expression of Notch 4 both in PBM and SPL as seen by flow cytometry and qPCR. Additionally, anti-Notch 1 treatment stimulated Notch transcription factors HES and HEY. Both PBM and SPL cultured OCPs from anti-Notch 1 treated mice produced a higher number of large TRAP+ osteoclasts, doubling the area covered with osteoclasts in the latter compared to untreated mice. Increased osteoclastogenesis in vitro was further confirmed by an increased expression of osteoclast differentiation genes in the treated group.Conclusion:Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo anti-Notch 1 neutralizing antibodies enhanced osteoclastogenesis in CIA model, implying an inhibitory role of Notch 1 signaling in osteoclast differentiation. As Notch 2 expression is increased on OCPs of arthritic mice, we next plan to determine the effects of Notch 2 neutralization on osteoclast activity and arthritis severity.References:[1]Ikić Matijašević M, Flegar D, Kovačić N, Katavić V, Kelava T, Šućur A, et al. Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis. Clin Exp Immunol. 2016;186(3):321–35.[2]Šućur A, Filipović M, Flegar D, Kelava T, Šisl D, Lukač N, et al. Notch receptors and ligands in inflammatory arthritis – a systematic review. Immunology Letters 2020 Vol. 223, p. 106–14.Acknowledgements:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, UIP-2017-05-1965 and DOK-2018-09-4276.Disclosure of Interests:None declared.

scholarly journals Effects of Titanium Surfaces on the Developmental Profile of Monocytes/Macrophages

2014 ◽  
Vol 25 (2) ◽  
pp. 96-103 ◽  
Author(s):  
Camilla Christian Gomes Moura ◽  
Darceny Zanetta-Barbosa ◽  
Paula Dechichi ◽  
Valessa Florindo Carvalho ◽  
Priscilla Barbosa Ferreira Soares
Keyword(s):  
Cell Viability ◽  
Colorimetric Assay ◽  
Initial Period ◽  
Critical Role ◽  
Surface Characteristics ◽  
Th2 Cytokine ◽  
Titanium Surfaces ◽  
Control Surfaces ◽  
Immunoenzymatic Assays

Due to the critical role of monocytes/macrophages (Mϕ) in bone healing, this study evaluated the effects of bio-anodized, acid-etched, and machined titanium surfaces (Ti) on Mϕ behavior. Cells were separated from whole human blood from 10 patients, plated on Ti or polystyrene (control) surfaces, and cultured for 72 h. At 24, 48 and 72 h, cell viability, levels of IL1β, IL10, TNFα, TGFβ1 inflammatory mediators, and nitric oxide (NO) release were analyzed by mitochondrial colorimetric assay (MTT assay) and immunoenzymatic assays, respectively. Real-time PCR was used to verify the expression of TNFα and IL10 at 72 h. The data were subjected to a Kruskal-Wallis analysis. IL1β, TNFα and TGFβ1 release were not significantly different between the Ti surfaces (p>0.05). The presence of NO and IL10 was not detected in the samples. Cell viability did not differ between the samples cultivated on Ti and those cultivated on control surfaces, except at 24 h (p=0.0033). With respect to the mediators evaluated, the surface characteristics did not induce a typical Th1 or Th2 cytokine profile, although the cell morphology and topography were influenced by the Ti surface during the initial period.

scholarly journals Bufei Qingyu Granules Inhibit the Development of Systemic Sclerosis via Notch-1/Jagged-2 Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-16
Author(s):  
Minhui Su ◽  
Fang Tian ◽  
Bingchen Ouyang ◽  
Xiaoyu Wu ◽  
Feng Guo ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Notch Signaling ◽  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Smooth Muscle Actin ◽  
Notch Signaling Pathway ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Notch 1 ◽  
Bioinformatics Analyses

Systemic sclerosis (SSc) is a rare chronic autoimmune disorder, mainly characterized by skin sclerosis. In this study, Bufei Qingyu Granules (BQG), a Chinese herbal formula, was used to treat SSc. To better understand the effects and molecular mechanisms of BQG, we successfully established a Bleomycin- (BLM-) induced SSc mouse model, and the mice were treated by BQG. Meanwhile, transcriptomic and bioinformatics analyses were conducted on those samples. As a result, we visually showed that BQG ameliorated the overall health of mice, including body weight, spleen, and thymus index. Thus, it also significantly alleviated inflammation presented by Chemokine (C-X-C motif) ligand 2 (Cxcl2), vasculopathy characterized by α-smooth muscle actin (α-SMA), and fibrotic changes elaborated by not only pathological images, but also the hydroxyproline (HYP) content. After testing by transcriptomic analysis, Cxcl2, Synaptosomal-associated protein 25 (Snap25), and Eukaryotic translation initiation factor 3, and subunit J2 (Eif3j2) which were differentially expressed genes, were verified, so that the data were credible. We further found that BQG could regulate Notch signaling pathway by significantly decreasing both mRNA and protein expression levels of Notch-1 and Jagged-2. Hence, this study demonstrated that BQG could ameliorate the sclerotic skin in mice model involved in inflammation, vascular changes, and fibrosis effects, which was partly mediated by Notch signaling pathway.

Use of a mass balance model for developing guidelines for treatment plant recycle streams

2001 ◽  
Vol 1 (4) ◽  
pp. 169-176
Author(s):  
K.H. Carlson ◽  
W.H. Bellamy
Keyword(s):  
Treatment Effectiveness ◽  
Treatment Plant ◽  
Significant Risk ◽  
Balance Model ◽  
Sludge Dewatering ◽  
Adequate Treatment ◽  
Materials Balance ◽  
Treatment Conditions ◽  
The Impact ◽  
Plant Treatment

Recycling treatment, plant waste streams has become an important issue and the EPA is currently developing a rule for controlling and potentially limiting these streams. The impact to the overall treatment process and the relative risk of various recycle streams can be evaluated with a materials balance model of a treatment plant. A steady-state materials balance model was developed and applied to the recycle of backwash waste water, clarifier sludge supernatant and sludge dewatering supernatant. Recycling backwash water reduced the plant treatment effectiveness from 3.0 log to 2.95 log removal when the recycle stream was treated (0.5 log removal) and 2.84 log removal when not treated. Recycling clarifier sludge supernatant resulted in a reduction of performance from 3.0 log to 2.78 log removal with adequate treatment (0.5 log removal) and 1.95 log removal with inadequate treatment (10% removal). The model was used to identify vulnerable treatment conditions. Conventional treatment with a poorly operated or upset clarifier was identified as a significant risk with the overall treatment effectiveness decreasing from 3.0 to 2.3 log removal.

scholarly journals Notch-1 Signaling Modulates Macrophage Polarization and Immune Defense against Mycobacterium avium paratuberculosis Infection in Inflammatory Diseases

2020 ◽  
Vol 8 (7) ◽  
pp. 1006 ◽  
Author(s):  
Esra’a Keewan ◽  
Saleh A. Naser
Keyword(s):  
Inflammatory Response ◽  
Notch Signaling ◽  
Mycobacterium Avium ◽  
Macrophage Polarization ◽  
Immune Defense ◽  
Notch 1 ◽  
Macrophage Response ◽  
Mycobacterium Avium Paratuberculosis ◽  
Vitro Effect

Despite the extensive research on Notch signaling involvement in inflammation, its specific role in macrophage response in autoimmune disease and defense mechanisms against bacterial infection, such as Mycobacterium avium paratuberculosis (MAP), remains unknown. In this study, we investigated the molecular role of Notch-1 signaling in the macrophage response during MAP infection. In particular, we measured the in vitro effect of MAP on Notch-1 signaling and downstream influence on interleukin (IL)-6 and myeloid cell leukemia sequence-1 (MCL-1) and consequent cellular apoptosis, MAP viability, and macrophage polarization. Overall, the data show significant upregulation in Notch-1, IL-6, and MCL-1 in MAP-infected macrophages, parallel with a decrease in apoptosis and elevated pro-inflammatory response in these infected cells. On the contrary, blocking Notch signaling with γ-secretase inhibitor (DAPT) decreased MAP survival and burden, increased apoptosis, and diminished the pro-inflammatory response. In particular, the treatment of infected macrophages with DAPT shifted macrophage polarization toward M2 anti-inflammatory phenotypic response. The outcome of this study clearly demonstrates the critical role of Notch signaling in macrophage response during infection. We conclude that MAP infection in macrophages activates Notch-1 signaling and downstream influence on IL-6 which hijack MCL-1 dependent inhibition of apoptosis leading to its chronic persistence, and further inflammation. This study supports Notch-1 signaling as a therapeutic target to combat infection in autoimmune diseases such as Crohn’s disease and Rheumatoid Arthritis.

Select γ-Secretase Inhibitors Induce Apoptosis in Pre-B ALL Cells and Disrupt the Balance Between Constitutive Notch Signaling and Repression.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1917-1917
Author(s):  
Bridget S. Wilson ◽  
Xiangbing Meng ◽  
Tomas Mazel ◽  
Cheryl L. Willman ◽  
Susan Atlas ◽  
...  
Keyword(s):  
Cell Death ◽  
Notch Signaling ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Caspase Activation ◽  
Notch 1 ◽  
Activating Mutations ◽  
Important Group ◽  
Notch Receptors ◽  
Activation Status

Abstract Several γ secretase inhibitors (GSIs) were tested for the ability to induce apoptosis in precursor B acute lymphoblastic leukemia (pre-B ALL) cells. Of five GSI’s tested, treatment with two compounds resulted in effective killing of both pre-B lymphoblasts and cells from multiple pre-B ALL lines. Since Notch receptors represent an important group of γ secretase targets, we evaluated expression and activation status of Notch receptors in CD19+ lymphoblasts from pediatric pre-B ALL patients, as well as cultured pre-B ALL cells. We found that, unlike T-ALL where activating mutations are common, pre-B ALL cells appear to drive constitutive Notch signaling through autocrine signals. Blasts from 11 patients expressed 3 Notch receptors and multiple Notch counter-ligands. Expression of Notch pathway genes was also confirmed by microarray analysis of genes expressed in 207 children with high risk B precursor ALL. GSI treatment of pre-B ALL cells led to dephosphorylation of AKT and Foxo3, Bim expression and caspase activation. GSI treatment also blocked cleavage of Notch 1 and 2 to their active forms and inhibited expression of Notch targets, Hey2 and Myc. In contrast, increased expression of Hes1 and Hey1 was correlated with GSI-induced loss of the co-repressor, SMRT. GSI treatment appears to induce precursor B cell death by disrupting the balance between constitutive Notch signaling and repression.

Prevalence and Risk Factors of Monoclonal Proteinemia in Thai Population.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1416-1416
Author(s):  
Phandee Watanaboonyongcharoen ◽  
Thanyaphong Na Nakorn ◽  
Ponlapat Rojnuckarin ◽  
Panisinee Lawasut ◽  
Tanin Intragumtornchai
Keyword(s):  
Risk Factors ◽  
Complete Blood Count ◽  
Conflicts Of Interest ◽  
Demographic Data ◽  
Significant Risk ◽  
Chemical Exposure ◽  
Blood Chemistry ◽  
Serum Samples ◽  
Thai Population ◽  
History Of

Abstract Abstract 1416 Poster Board I-439 Background: Monoclonal gammopathy of undetermined significance (MGUS) is associated with progression to multiple myeloma and related hematologic malignancies at the rate of 1% per year in western population. Reliable information on prevalence, risk factors and natural history of MGUS in general population are necessary for designing an early detection strategy for myeloma in Thailand. Patients and methods: The study was performed in subjects, 50 years of age or older, in Bangkok, one nearby province and 2 remote provinces of Thailand. The demographic data and suspected risk factor history were collected by questionnaires. Complete blood count as well as blood chemistry were done to exclude underlying hematologic and/or systemic conditions. Serum monoclonal proteins were detected using high-resolution gel electrophoresis. Results: Serum samples were obtained from 3,261 participants. There were 1,105 males (33.9%) and 2,156 females (66.1%). The median age was 57. Abnormal protein electrophoresis findings were detected in 76 samples (prevalence 2.3%, 95% confidence interval [CI] 1.8% - 2.8%) showing small M-spikes at gamma-globulin region in 50 (1.5%) or at beta-globulin region in 25 (0.8%) or hypogammaglobulinemia in 1 case (0.03%). The prevalence of MGUS in subjects less than 60, 60-69 and 70 yrs or more was 2.0% (41/1975), 2.6% (22/851) and 3.0% (13/435), respectively. Using multivariate analysis, presence of MGUS was strongly associated with history of drug abuse (odd ratio 4.63, 95%CI 1.14-22.08) and current residences outside Bangkok (odd ratio 2.30, 95%CI 1.18-4.79). Radiation and chemical exposure, hair and nail-coloring products and pesticides were not statistically significant risk factors in our population. Conclusions: The overall prevalence of MGUS in Thai population was 2.3%, lower than those of western countries but comparable to what reported from Japan and Taiwan. Disclosures: No relevant conflicts of interest to declare.

Myeloma Cell Survival and Importance of Crosstalk Between Notch1-Jagged2 and CD28-B7 Pathways In Dendritic Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1741-1741
Author(s):  
Chandana Koorella ◽  
Jayakumar Nair ◽  
Louise Carlson ◽  
Megan Murray ◽  
Cheryl H Rozanski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Notch Signaling ◽  
Cell Biology ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Negative Regulator ◽  
Soluble Form ◽  
Myeloma Cells ◽  
Notch1 Signaling

Abstract Abstract 1741 Multiple myeloma is a neoplasm of bone marrow resident plasma cells characterized by critical interactions between myeloma cells and bone marrow stromal cells. This interaction leads to production of IL-6, an important factor in myeloma cell biology. However, the molecular and cellular components involved in myeloma induced IL-6 production remain largely uncharacterized. While at the cellular level, dendritic cells (DC)-expressing CD80/CD86 (collectively called B7, ligands with short cytoplasmic tails and signaling partners of CD28 expressed on myeloma cells) - in the bone marrow microenvironment have been implicated as being an important component, at the molecular level the CD28-B7 and Notch1-Jagged2 pathways were separately implicated by us (in DC) and others in myeloma induced IL-6 production. Although Notch signaling leading to IL-6 production in DC is well understood, the mechanism of “backsignaling” via B7 is largely uncharacterized. To better understand downstream B7 signaling leading to IL-6 production, DC were stimulated with CD28-Ig (a soluble form of CD28 which mimicks myeloma cell-bound CD28) in the presence or absence of an inhibitor of Notch signaling, gamma secretase inhibitor (GSI). DC treated with CD28-Ig alone produced significantly (p< 0.001) higher levels of IL-6 when compared to DC treated with CD28-Ig and GSI. GSI specifically targeted Notch signaling as observed by decreased expression of Notch gene targets: Hes-1 (2 fold decrease) and Deltex-4 (4 fold decrease). Also, decreased IL-6 levels in presence of GSI were not due to the decrease in B7 expression on DC. To specifically implicate the importance of Notch1 and Jagged2, we blocked Notch1 signaling using blocking antibodies and observed a similar decrease in IL-6 production upon blocking Notch1 signaling. Our results suggest that CD28 mediated IL-6 production is dependent on Notch1 signaling and crosstalk between the Notch1-Jagged2 and CD28-B7 pathways leads to IL-6 production by DC. The model of crosstalk between CD28-B7 and Notch1-Jagged2 pathways was also observed in murine bone marrow derived dendritic cells (BMDC), where a significant (p<0.001) down regulation of IL-6 was observed upon blocking Notch signaling. One possible mechanism of crosstalk involves direct effect of B7 crosslinking by CD28-Ig on Notch expression/signaling leading to increase in IL-6 production. We tested for this possibility in DC and found no significant change in Notch expression/signaling. We thus hypothesized that the mechanism of crosstalk involves molecules downstream to Notch and/or B7. Notch signaling has been reported to be involved in the regulation of PTEN (a negative regulator of the PI3K/Akt pathway). Previous studies have also shown the importance of FoxO3a-a transcription factor tightly regulated by Akt- in regulating IL-6 production in BMDC upon B7 crosslinking. We therefore tested the possible involvement of PTEN (molecule downstream of Notch signaling), Akt and FoxO3a (molecules downstream of B7) in crosstalk between the two pathways aforementioned by testing the effect of GSI on their regulation at the protein level. We observed an approximate 2 fold decrease in phospho-PTEN/PTEN ratio in DC treated with GSI and remained so even after B7 crosslinking at an early time point (15 min. post CD28-Ig treatment.) Further, phospho-Akt/Akt ratio decreased by 1.6 fold in DC treated with both GSI and CD28-Ig compared to CD28-Ig alone at 30 min. We therefore hypothesize a model of crosstalk involving Notch mediated regulation of PTEN leading to IL-6 production via regulation of Akt and possibly FoxO3a upon B7 crosslinking. Interestingly enough “backsignaling” via B7 in myeloma-induced IL-6 production seems to involve molecules well characterized in CD28 signaling of T-cells. Targeting IL-6 induced by crosstalk between these two pathways prompts not only clinical evaluation to improve MM patient outcome but also extends to advancing knowledge in T-cell and normal plasma cell biology as well. Disclosures: No relevant conflicts of interest to declare.

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