Activation Of Notch Signaling Pathway Research Articles

SRCAP is involved in porcine reproductive and respiratory syndrome virus activated Notch signaling pathway

ABSTRACT Porcine reproductive and respiratory syndrome viru s (PRRSV) is the cause of porcine reproductive and respiratory syndrome (PRRS); a disease of pigs, which results in great economic losses in the pork industry. The non-structural protein 4 (Nsp4), a 3C-like serine protease responsible for most non-structural protein processing, plays an essential role in PRRSV infection. We used label-free quantitative proteomics to elucidate the Nsp4 interactome and SRCAP was identified as one of the interactors. SRCAP facilitated PRRSV infection by activating non-canonical Notch signaling. The ATPase I–IV domain in SRCAP and the 122 VITEA 126 in Nsp4 were identified as the interacting sites. The infection of recovered mutant rTA-12/5A ( 122 AAAAA 126 ) could not activate Notch signaling. The results indicated that 122 VITEA 126 in Nsp4 were key sites to determine the function of SRCAP and their interaction. A function of Nsp4 in activating the Notch signaling pathway was discovered. Block Notch signaling pathway could inhibit PRRSV infection both in vitro and in vivo which may lead to the development of novel therapeutic antiviral strategies. IMPORTANCE In the present study, the interactome of the NSP4 originating from PRRSV was studied and SRCAP was confirmed as one of the interactors. Mechanism study showed the interaction of Nsp4 and SRCAP was found to facilitate PRRSV infection by activating non-canonical Notch signaling. ATPase Ⅰ–Ⅳ domain in SRCAP and the 122 VITEA 126 in Nsp4 were identified as the interacting sites that demined the activating of Notch signaling. Block Notch signaling pathway could inhibit PRRSV infection in vitro and in vivo which may be a new target for antiviral drug development.

Effect of Sortilin1 on promoting angiogenesis and systemic metastasis in hepatocellular carcinoma via the Notch signaling pathway and CD133

Hepatocellular carcinoma (HCC) is known to be lethal disease. However, its prognosis remains poor, primarily because the precise oncogenic mechanisms underlying HCC progression remain elusive, thus hampering effective treatment. Here, we aimed to identify the potential oncogenes in HCC and elucidate the underlying mechanisms of their action. To identify potential candidate genes, an integrative analysis of eight publicly available genomic datasets was performed, and the functional implications of the identified genes were assessed in vitro and in vivo. Sortilin 1 (SORT1) was identified as a potential candidate oncogene in HCC, and its overexpression in HCC cells was confirmed by analyzing spatial transcriptomic and single-cell data. Silencing SORT1 in Huh-7 and Hep3B cells significantly reduced HCC progression in vitro and in vivo. Functional analyses of oncogenic pathways revealed that SORT1 expression regulated the Notch signaling pathway activation and CD133 expression. Furthermore, analysis of epigenetic regulation of the candidate gene and its clinical implications using The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC) and our HCC cohort (AJOU_HCC cohort) data demonstrated an inverse correlation between the methylation status of the SORT1 promoter region, specifically at the cg16988986 site, and SORT1 mRNA expression, indicating the epigenetic regulation of SORT1 in HCC. In addition, the distinct methylation status of cg16988986 was significantly associated with patient survival. In conclusion, SORT1 plays a pivotal role in HCC by activating the Notch signaling pathway and increasing CD133 expression. These findings suggest SORT1 as a promising therapeutic target for HCC.

SIRT5 suppresses the trophoblast cell proliferation, invasion, and migration to promote preeclampsia via desuccinylating HOXB3.

Preeclampsia (PE) is a pregnancy-specific syndrome with increasing maternal and perinatal morbidity and mortality. Succinylation, a post-translational modification event, has been found in various diseases. However, the role of succinylation in PE has not been explored. This study aimed to investigate the effect of succinylation on PE and the underlying mechanisms. Thirty-two PE patients and 32 normal pregnancy volunteers were recruited. Human extravasated trophoblast cells (HTR-8/SVneo) were used in in vitro study. RT-qPCR was performed to detect the expression of succinylation-related mRNAs. The cell proliferation, invasion, and migration were assessed using cell counting kit-8, ethynyldeoxyuridine, transwell, and wound healing assays. Co-immunoprecipitation and dual-luciferase reporter assays were performed to analyze the interaction between sirtuin (SIRT)5 and homeobox box 3 (HOXB3). SIRT5 was increased in the placental tissues of PE patients. SIRT5 inhibition increased cell proliferation, invasion, and migration in HTR-8/SVneo cells. Mechanistic investigations indicated that HOXB3 was a downstream regulatory target of SIRT5-mediated desuccinylation. Rescue experiments further verified that silencing of HOXB3 inhibited cell proliferation, invasion, and migration. Additionally, HOXB3 deficiency reversed the activation of the Notch and β-catenin signaling pathway induced by SIRT5 inhibition. SIRT5 inhibited the trophoblast cell proliferation, invasion, and migration to promote PE through suppressing Notch and β-catenin signaling pathway activation via desuccinylating HOXB3.

LncRNA PCAT6 promotes the occurrence of laryngeal squamous cell carcinoma via modulation of the miR-4731-5p/NOTCH3 axis.

Laryngeal squamous cell carcinoma (LSCC) is one of the most aggressive cancers that affect the head and neck region. Recent researches have confirmed that long non-coding RNAs (lncRNAs) present an emerging role in diversiform diseases including cancers. Prostate cancer-associated ncRNA transcript 6 (PCAT6) is an oncogene in lung cancer, cervical cancer, colon cancer and gastric cancer, but its role in LSCC is still unknown. In the current study, we attempted to figure out the role of PCAT6 in LSCC. RT-qPCR was to analyze PCAT6 expression in LSCC cells. Functional assays were to uncover the role of PCAT6 in LSCC. Mechanism assays were to explore the regulatory mechanism behind PCAT6 in LSCC. PCAT6 exhibited higher expression in LSCC cells and PCAT6 strengthened cell proliferation and inhibited cell apoptosis. Furthermore, lncRNA PCAT6 modulated notch receptor 3 expression and activated NOTCH signaling pathway via serving as a sponge for miR-4731-5p. Taken together, lncRNA PCAT6 was identified as an oncogene in LSCC, which revealed that PCAT6 might be used as potential therapeutic target for LSCC.

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.

Activation of Notch Signaling Pathway is involved in Extracellular Matrix Degradation in human induced pluripotent stem cells chondrocytes induced by HT-2 toxin

Notch signaling regulates cartilage formation and homeostasis. Kashin-Beck Disease (KBD), an endemic osteochondropathy, is characterized by severe cartilage degradation. The etiology of KBD is related to the exposure of HT-2 toxin, a mycotoxin and primary metabolite of T-2 toxin. This study aims to explore the role of HT-2 toxin in the Notch signaling regulation and extracellular matrix (ECM) metabolism of hiPSCs-Chondrocytes. Immunohistochemistry and qRT-PCR were employed to investigate the expression of Notch pathway molecules in KBD articular cartilage and primary chondrocytes. hiPSCs-Chondrocytes, derived from hiPSCs, were treated with 100 ng/mL HT-2 toxin and the γ-secretase inhibitor (DAPT) for 48h, respectively. The markers related to the Notch signaling pathway and ECM were assessed using qRT-PCR and Western blot. Notch pathway dysregulation was prominent in KBD cartilage. HT-2 toxin exposure caused cytotoxicity in hiPSCs-Chondrocytes, and activated Notch signaling by increasing the mRNA and protein levels of NOTCH1 and HES1. HT-2 toxin also upregulated ECM catabolic enzymes and downregulated ECM components (COL2A1 and ACAN), indicating ECM degradation. DAPT-mediated Notch signaling inhibition suppressed the mRNA and protein level of ADAMTS5 expression while enhancing ECM component expression in hiPSCs-Chondrocytes. This study suggests that HT-2 toxin may induce ECM degradation in hiPSCs-Chondrocytes through activating Notch signaling.

Clinical significance of PNO1 as a novel biomarker and therapeutic target of hepatocellular carcinoma.

The RNA-binding protein PNO1 plays an essential role in ribosome biogenesis. Recent studies have shown that it is involved in tumorigenesis; however, its role in hepatocellular carcinoma (HCC) is not well understood. The purpose of this study was to examine whether PNO1 can be used as a biomarker of HCC and also examine the therapeutic potential of PNO1 knockout for the treatment of HCC. PNO1 expression was upregulated in HCC and associated with poor prognosis. PNO1 expression was positively associated with tumour stage, lymph node metastasis and poor survival. PNO1 expression was significantly higher in HCC compared to that in fibrolamellar carcinoma or normal tissues. Furthermore, HCC tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53. PNO1 knockout suppressed cell viability, colony formation and EMT of HCC cells. Since activation of Notch signalling pathway promotes HCC, we measured the effects of PNO1 knockout on the components of Notch pathway and its targets. PNO1 knockout suppressed Notch signalling by modulating the expression of Notch ligands and their receptors, and downstream targets. PNO1 knockout also inhibited genes involved in surface adhesion, cell cycle, inflammation and chemotaxis. PNO1 knockout also inhibited colony and spheroid formation, cell migration and invasion, and markers of stem cells, pluripotency and EMT in CSCs. Overall, our data suggest that PNO1 can be used as a diagnostic and prognostic biomarker of HCC, and knockout of PNO1 by CRISPR/Cas9 can be beneficial for the management of HCC by targeting CSCs.

Natural killer cell-based signature: Prognostic analysis in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSC) is a challenging cancer with significant clinical implications. Natural killer (NK) cells have emerged as important players in tumor immunosurveillance, yet their role and potential as prognostic biomarkers in HNSC remain unclear. Quantitative analysis using multiple algorithms identified FCRL1, KIR3DL2 and ZNF541 as molecules significantly associated with local NK cell infiltration and patient survival. A prognostic model based on these molecules demonstrated robust predictive performance. Analysis of high- and low-risk patient groups revealed distinct differences in the tumor microenvironment, indicating an inhibitory immune microenvironment in high-risk patients. Notably, low-risk patients exhibited potential sensitivity to immunotherapy and showed favorable responses to specific drugs such as axitinib, methotrexate, rapamycin and vorinostat. NK cells, important effectors of the innate immune response, were found to play a crucial role in HNSC immunity. The present study provides valuable insights into the correlation between FCRL1, KIR3DL2, ZNF541 and NK cell infiltration, paving the way for future investigations into their roles in HNSC. Activation of NOTCH signaling, MYC targets, DNA repair, E2F targets, epithelial-mesenchymal transition, G2M checkpoint and mitotic spindle pathways in high-risk patients suggests their involvement in disease progression and poor prognosis. The present study reveals the significance of NK cells in HNSC and their potential as prognostic biomarkers. The CFKZ score offers a promising approach for predicting patient outcomes and guiding personalized treatment decisions in HNSC. These findings contribute to our understanding of HNSC immunobiology and hold implications for precision medicine in HNSC management.

Activation of Notch-1 signaling pathway in macrophages to secrete PD-L1 and regulate cytotoxicity of CAR-T cells in diffuse large B-cell lymphoma.

To investigate the mechanism of action of the Notch-1/IRE1/XBP1s signaling pathway in diffuse large B-cell lymphoma (DLBCL). The expressions of relevant proteins were detected by Western blotting. The effect of myeloid-specific knockout of Notch-1 on lymphoma progression was observed by mouse tumor transplantation and imaging. The apoptosis of chimeric antigen receptor T-cell therapy (CAR-T) cells were detected by flow cytometry, and the proliferation of CAR-T cells was detected by wound healing assay and cell counting kit-8 (CCK8) assay. Lymphoma cells mediated the Notch-1 signaling pathway in bone marrow-derived macrophages and promoted the activation of STAT3 and STAT6 in bone marrow-derived macrophages. Myeloid-specific knockout of Notch-1 could inhibit the progression of lymphoma. Lymphoma cells enhanced the expression of p-PERK, p-IRE1α, ATF6, IL-6, IL-4, p-AKT, CD9, CD63 and PD-L1 in bone marrow-derived macrophages by mediating the Notch-1 signaling pathway. Knockout of Notch-1 in macrophages alleviated, to some extent, the suppression of killing activity of CAR-T cells, while activation of Notch-1 in macrophages inhibited proliferation and promoted apoptosis of CAR-T cells. The PD-L1 antibody significantly restored the cytotoxicity and proliferation of CAR-T cells, and inhibited their apoptosis. Activation of the Notch-1/IRE1/XBP1s signaling pathway in myeloid macrophages promotes the secretion of IL-6 and IL-4 as well as PD-L1, thereby inhibiting the activity and proliferation of CAR-T cells and promoting their apoptosis.

A comprehensive integrated gene network construction to explore the essential role of Notch 1 in lung adenocarcinoma (LUAD)

The heterogeneous biological landscape of non-small cell lung cancer (NSCLC) is largely attributed to the activation of Notch signalling pathway. Among the Notch family transmembrane proteins, neurogenic locus notch homolog protein1 (NOTCH1) is a putative oncogene in NSCLC which activates the pathway as negative prognostic factor. This study aims to explore integrated network approach in lung adenocarcinoma (LUAD) especially linked to the notch pathway and its receptors. Our gene set enrichment analysis reveals the key Notch pathway genes are predominantly down regulated in LUAD. There were 675 genes with a total of 6517 functional interactions and 6 densely connected clusters of 38 miRNAs, 84 transcription factors with 156 edges identified through network construction. Here we report five key genes namely NOTCH1, CDH1, ERBB2, GAPDH and COL1A1 significantly enriched in Notch pathway which are further validated through the KM plot, box plots, stage plots and TIMER analysis. In addition, the NOTCH1 receptor is strongly linked to the immune checkpoint inhibitor CD274 (PD-L1) and can be considered as prognostic marker and tumour suppressor gene in LUAD which surely provide the basis for early diagnosis and futuristic immunotherapeutic targets for LUAD. Communicated by Ramaswamy H. Sarma

DTL promotes head and neck squamous cell carcinoma progression by mediating the degradation of ARGLU1 to regulate the Notch signaling pathway

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with a high incidence in squamous epithelium. The E3 ubiquitin ligase DTL is a component of the CRL4A complex and is widely involved in tumor progression. We aimed to analyze the role of DTL in HNSCC and to explore its mechanism of action. Through clinical analysis, we found that DTL is upregulated in HNSCC tissues and is associated with the tumor microenvironment and poor survival in patients. Through gain-of-function and loss-of-function assays, we showed that DTL promotes cell proliferation and migration in vitro and tumor growth in vivo. Mass spectrometry analysis and immunoprecipitation assays showed that DTL interacts with ARGLU1 to promote K11-linked ubiquitination-mediated degradation of ARGLU1, thereby promoting the activation of the CSL-dependent Notch signaling pathway. Furthermore, siARGLU1 blocks the inhibitory effects of DTL knockdown on HNSCC cells. In this study, we showed that DTL promotes HNSCC progression through K11-linked ubiquitination of ARGLU1 to activate the CSL-dependent Notch pathway. These findings identify a promising therapeutic target for HNSCC.

GRWD1 Over-Expression Promotes Gastric Cancer Progression by Activating Notch Signaling Pathway via Up-Regulation of ADAM17.

Glutamate-rich WD repeat containing 1 (GRWD1) is over-expressed in a variety of malignant tumors and is considered to be a potential oncogene. However, its mechanism of action in gastric cancer (GC) is still unclear. Data analysis, Immunohistochemistry, and Western Blot (WB) were performed to verify the expression of GRWD1 in GC and para-cancerous tissues. The association between GRWD1 expression and tumor size, tissue differentiation, lymph node metastasis, TNM stage, and prognosis was analyzed according to the high and low expression levels of GRWD1. The relationship between GRWD1 and Notch pathway was verified by data analysis and WB. The effects of GRWD1 on the proliferation, migration, and invasion of GC cells were verified by cell proliferation, migration, and invasion assays. We confirmed that the high expression of GRWD1 promoted the proliferation of GC cells in vivo through the tumor formation assay in nude mice. The expression of GRWD1 was higher in GC tissues than in para-cancerous tissues, and its expression was positively correlated with tumor size, lymph node metastasis, and TNM stage, but negatively correlated with differentiation grade and prognosis. GRWD1 over-expression increased ADAM metallopeptidase domain 17 (ADAM17) expression and promoted Notch1 intracellular domain (NICD) release to promote GC cell proliferation, migration, and invasion in vitro. Results from animal studies have shown that high GRWD1 expression could promote GC cell proliferation in vivo by activating the Notch signaling pathway. GRWD1 promotes GC progression through ADAM17-dependent Notch signaling, and GRWD1 may be a novel tumor marker and therapeutic target.

A hsa_circ_001726 axis regulated by E2F6 contributes to metastasis of hepatocellular carcinoma

BackgroundCircRNAs participate in the development of hepatocellular carcinoma (HCC). This work aims to explore the key tumor promoting circRNA as a gene therapy target.MethodsThe differentially expressed gene circRNAs in HCC tumor tissues was identified by mining GSE121714 dataset. EdU staining, wound healing, transwell invasion assay, TUNEL staining and western blotting examined proliferation, migration, invasion, apoptosis and epithelial mesenchymal transition (EMT). Xenograft mouse model and orthotopic transplantation tumor mouse model were constructed to verify the role of hsa_circ_001726 in growth and metastasis of HCC. The relationship among CCT2, E2F6, hsa_circ_001726, miR-671-5p and PRMT9 was identified by RNA-fluorescence in situ hybridization, luciferase reporter assay and RNA Immunoprecipitation.ResultsEleven differentially expressed circRNAs were found in HCC tumors. Among them, hsa_circ_001726 was highly expressed in HCC tumors and cells, which was transcribed from CCT2. As a transcription factor of CCT2, E2F6 knockdown inactivated CCT2 promoter and reduced hsa_circ_001726 expression. Moreover, hsa_circ_001726 elevated PRMT9 expression by sponging miR-671-5p, and then activated Notch signaling pathway. Additionally, hsa_circ_001726 deficiency repressed malignant phenotypes of HCC cells, including proliferation, migration, invasion, EMT and apoptosis. In vivo, hsa_circ_001726 deficiency reduced tumor growth and lung metastasis of HCC in xenograft mouse models and orthotopic transplantation tumor mouse models.ConclusionHsa_circ_001726 functioned as an oncogene in HCC, which was derived from CCT2 and regulated by E2F6. Hsa_circ_001726 elevated PRMT9 expression by sponging miR-671-5p, and then activated Notch signaling pathway, thereby accelerating malignant phenotypes of HCC. Therefore, targeting hsa_circ_001726 may be a new avenue for HCC treatment.

KMT2D mutations promoted tumor progression in diffuse large B-cell lymphoma through altering tumor-induced regulatory T cell trafficking via FBXW7-NOTCH-MYC/TGF-β1 axis.

Histone methyltransferase KMT2D is one of the most frequently mutated genes in diffuse large B-cell lymphoma (DLBCL) and has been identified as an important pathogenic factor and prognostic marker. However, the biological relevance of KMT2D mutations on tumor microenvironment remains to be determined. KMT2D mutations were assessed by whole-genome/exome sequencing (WGS/WES) in 334 patients and by targeted sequencing in 427 patients with newly diagnosed DLBCL. Among all 761 DLBCL patients, somatic mutations in KMT2D were observed in 143 (18.79%) patients and significantly associated with advanced Ann Arbor stage and MYC expression ≥ 40%, as well as inferior progression-free survival and overall survival. In B-lymphoma cells, the mutation or knockdown of KMT2D inhibited methylation of lysine 4 on histone H3 (H3K4), downregulated FBXW7 expression, activated NOTCH signaling pathway and downstream MYC/TGF-β1, resulting in alterations of tumor-induced regulatory T cell trafficking. In B-lymphoma murine models established with subcutaneous injection of SU-DHL-4 cells, xenografted tumors bearing KMT2D mutation presented lower H3K4 methylation, higher regulatory T cell recruitment, thereby provoking rapid tumor growth compared with wild-type KMT2D via FBXW7-NOTCH-MYC/TGF-β1 axis.

LINC00460 promotes neuroblastoma tumorigenesis and cisplatin resistance by targeting miR-149-5p/DLL1 axis and activating Notch pathway invitro and in vivo.

Long noncoding RNAs (lncRNAs) have been demonstrated to participate in neuroblastoma cisplatin resistance and tumorigenesis. LncRNA LINC00460 was previously reported to play a critical regulatory role in many cancer development. Nevertheless, its role in modulating neuroblastoma cisplatin resistance has not been explored till now. Cisplatin-resistant neuroblastoma cell lines were established by exposing neuroblastoma cell lines to progressively increasing concentrations of cisplatin for 6months. LINC00460, microRNA (miR)-149-5p, and delta-like ligand 1 (DLL1) mRNA expression was measured through RT-qPCR. The protein levels of DLL1, epithelial-to-mesenchymal transition (EMT) markers, and the Notch signaling-related molecules were measured via western blotting. The IC50 value for cisplatin, cell growth, metastasis and apoptosis were analyzed in cisplatin-resistant neuroblastoma cells. The binding between LINC00460 (or DLL1) and miR-149-5p was validated through dual-luciferase reporter assay. The murine xenograft model was established to perform in vivo assays. LINC00460 and DLL1 levels were elevated, while miR-149-5p level was reduced in cisplatin-resistant neuroblastoma cells. LINC00460 depletion attenuated IC50 values for cisplatin, weakened cell growth, metastasis, and EMT, and enhanced apoptosis in cisplatin-resistant neuroblastoma cells. Mechanically, LINC00460 sponged miR-338-3p to increase DLL1 level, thereby activating Notch signaling pathway. DLL1 overexpression antagonized LINC00460 silencing-induced suppression on neuroblastoma cell cisplatin resistance and malignant behaviors, while such effects were further reversed by treatment with DAPT, the inhibitor of Notch pathway. Additionally, LINC00460 knockdown further augmented cisplatin-induced impairment on tumor growth in vivo. LINC00460 contributes to neuroblastoma cisplatin resistance and tumorigenesis through miR-149-5p/DLL1/Notch pathway, providing new directions to improve the therapeutic efficacy of chemotherapy drugs applied in patients with neuroblastoma.

Truncating SPEN Mutations Highlight BN2-Subtype DLBCL with Aggressive Biology and Features of Immune Evasion

Introduction: Molecular classifications have been developed to characterize the heterogeneity of diffuse large B cell lymphoma (DLBCL), with activation of BCL6 and Notch signaling pathways acting as driving pathogenic features in C1/BN2 subtype tumors. Among C1/BN2 defining variants are truncating/inactivating SPEN mutations. SPEN regulates the Notch signaling pathway by initiating formation of a repressive complex that facilitates transcriptional repression of Notch target genes. Our group has identified SPEN mutations enriched in DLBCL cases failing to achieve event-free status at 24 months (EFS24). This contrasted with generally favorable outcomes in C1/BN2 tumors, suggesting a potential high-risk molecular feature with unexplored significance. Presenting an opportunity to expand underlying mechanisms and risk stratification in C1/BN2 tumors, we aimed to use a functional genomic approach, combined with clinical data, to characterize the alternative biology stemming from SPEN truncating mutations. Methods: FFPE tumor biopsies from newly diagnosed DLBCL cases (N=444) enrolled in the Mayo Clinic/Iowa Lymphoma Molecular Epidemiology Resource (MER) program were analyzed by WES (n=404) and RNA-seq (n=321). Biologic and clinical variables were annotated for all cases. CRISPR/Cas9 introduced truncating mutations to the endogenous SPEN locus in OCI-LY3 & SU-DHL-2 ABC-type human cell lines to establish models of SPEN depletion. Functional in vitro analysis utilized standard methods for RNA-seq, cell proliferation, western blot, and flow cytometry. Results: WES identified 29/404 ndDLBCL (7.2%) harboring SPEN mutations; n=17 missense and n=12 nonsense or frameshift insertions/deletions (truncating variants - [trunc]), the later considered as the focal point of our study. Against all cases, SPEN trunc cases displayed enrichment for high-risk factors including non-GCB COO (p=0.06, OR=3.7, 95% CI [0.9, 22.2]) and primary refractory disease (PTR; p=0.02, OR=5.0, 95% CI [1.05, 19.9]), and demonstrated inferior OS (p=0.06, HR=2.2), where a majority of clinical progression events occurred within 12 months of diagnosis. Leveraging RNA-seq, we observed 11 of 11 SPEN trunc cases exhibiting a double-hit negative gene expression signature, 9/11 cases with inflammatory or depleted microenvironment (LME), and 8/11 with a signature associating with intermediate/high risk of EFS24 failure. Applying the LymphGen algorithm, 5/12 SPEN trunc cases received the BN2 classification, observing co-occurring mutations with CD70 (p<0.01, OR=9.68), BRCA2 (p=0.03, OR=5.84), TET2 (p=0.02, OR=4.91), IRF4 (p=0.02, OR=7.27), and PTPRD (p<0.01, OR=8.31) genes, and were mutually exclusive with ARID1A, BCL2, EZH2, FAS, HLA-B, IRF8, and TMEM30A mutations ( Figure 1A). Isolating SPEN trunc against BN2 cases without SPEN variants (n=14), despite similar clustering of non-GCB COO (p=0.7), PTR was exclusively observed in SPEN trunc cases, accompanied again by inferior OS (p=0.07, HR=4.2; Figure 1B). In the OCI-LY3 SPEN depletion model, GSEA analysis from RNA-seq revealed activation of E2F (NES=2.7; p=9.4E-20) and MYC target genes (NES=2.3; p=4.1E-6), while both OCI-LY3 and SU-DHL-2 models featured depletion of antigen presentation genes (NES= -1.8, p=5.7E-4; NES= -2.2, p=3.8E-7, respectively). A decrease of MHC class II proteins (HLA-DR, HLA-DP) was confirmed by flow cytometry in both models (p<0.01). Hypothesizing loss of SPEN enhances reliance on the BCL6-HDAC3 axis featured in BN2 tumors, we treated both SPEN depletion models with a class-I HDAC inhibitor highly selective for HDAC3, BRD-3308, and indeed observed a 1.4 and 2.4-fold increase in sensitivity compared to WT cells (p=0.07, p<0.01, respectively). Significance: We find SPEN trunc mutations in poor acting DLBCL cases with clinical and biologic characteristics that diverge from C1/BN2 biology. The enhanced rate of clinical progression highlights urgency to better characterize these tumors. Our data suggests loss of SPEN regulatory mechanisms may act cooperatively with C1/BN2 biology to facilitate tumor immune evasion and drive progression through Notch-mediated pathways. Importantly, in vitro evidence shows SPEN deficient lymphoma cells are susceptible to targeted therapeutics against dependent pathways, providing rationale for advancing individualized care strategies for affected patients.

Effect of Valproic Acid on Promoting the Differentiation of Human Embryonic Stem Cells Into Cholangiocyte-Like Cells.

Cholangiocytes form a complex 3D network of bile ducts in the liver and contribute to liver function. The damage or destruction of cholangiocytes can lead to biliary diseases, and the shortage of cholangiocytes remains an obstacle for drug development targeting biliary diseases. Valproic acid (VPA) is a potent activator of Notch signaling pathway that is essential for cholangiocyte differentiation. Here, we report a VPA-based approach for cholangiocyte differentiation of human pluripotent stem cells. VPA activated Notch2 expression and upregulated HES-1, HEY-1, and Sox9 gene expression in hESC-derived hepatoblast. After 7 days treatment, VPA promoted successful differentiation of hepatoblast into cholangiocytes expressing cholangiocyte marker genes (AE2, AQP1, CFTR) and proteins (CK19 and CK7). In addition, the differentiated cholangiocytes formed bile duct-like structures after implantation into the spleen of NOD/SCID mice. Our results suggested that VPA can promote hESC differentiation to cholangiocyte-like cells. The induced cholangiocytes may serve as a potential cell source for both in vitro modeling and regenerative therapy of cholangiopathies. The findings can also support further development of small-molecule based differentiation protocols for cholangiocyte production.

CLL Cells within the Proliferation Centers of the Patient Lymph Nodes Are Enriched for Notch Signaling, Thus NOTCH a Viable Target for CLL Therapy

CLL cells within the Proliferation Centers of the Patient Lymph Nodes are enriched for Notch signaling, thus a Target for CLL Therapy. Shantaram S. Joshi, Michael Pitner, Matt Kling, Furqan Mehdi, Mamta Shukla, Ashima Shukla, Vipul Shukla, Nagendra Chaturvedi, Hamid Band , Javeed Iqbal The tissue microenvironment, particularly the lymph node (LN), plays a major role in regulating the leukemic progression and clinical heterogeneity of chronic lymphocytic leukemia (CLL). CLL cells proliferate in the patient lymph nodes, resulting in the formation of proliferation centers (PCs). The molecular basis of the CLL cell proliferation within PCs is not well known. Several reports have implicated the activated Notch signaling pathway in the pathogenesis of CLL. Using the IRF4 -/-Vh11 mouse model of CLL, we have previously shown that Notch signaling is indispensable for CLL development (Vipul Shukla et al, Oncotarget, 2016). Based on these studies, we hypothesized that CLL cell proliferation in the lymph node PCs involves activated NOTCH signaling. To this end, we have examined the expression levels of Notch signaling associated molecules namely IRF4, Notch 1, Notch 2, Nedd4, Fbxw7 and Itch1 on CLL cells within the patient lymph node proliferation centers (n=12) using immunohistochemistry and western blotting. Expression levels were scored based on the percentage of cells with positive staining: 5+ (>80%), 4+ (60-80%), 3+ (40-60%), 2+ (20-40%), 1+ (<20%) and 0 (No staining). IHC analyses showed that the expression levels of IRF4 to be 2+, Notch 1 was 4+, Notch 2 was -3 to 4+, Nedd4 was 1+, Fbxw7 was 3+ and ITCH1 was 1+. Western blotting of CLL cells from the lymph node proliferation centers (n=6) confirmed a similar pattern of expression of the Notch-associated genes. Together, these results validate the association of Notch signaling in CLL cells of the patient lymph node PCs similar to CLL cells from the IRF4 -/-Vh11 mouse model. Therefore, as a next logical step we treated the MEC-1 and OSU-CLL cells in culture and primary CLL cell from patients with NOTCH inhibitor RO4929097 (RO), a γ-secretase inhibitor for 24, 48 and 72 hours in vitro. There was a significant decrease in the expression NOTCH 1, NOTCH 2 in MEC-1, OSU-CLL and primary CLL cells determined using western blot technique. Also, we observed a significant decrease in proliferation of CLL cells from MEC-1 and OSU-CLL cell lines, following treatment with NOTCH inhibitor RO. In addition, NOTCH inhibitor combined with Bruton's Tyrosine Kinase (BTK) a member of the B-Cell Receptor signaling, showed a further significant decrease in the proliferation of CLL cells in culture. Together these experiments demonstrated that NOTCH targeting strategy is a viable therapy for aggressive CLL warranting further preclinical and clinical studies. This work was partially supported by bridge funding from the American Association of Hematology awarded to Dr. Runqing Lu, who passed away during the pursuit of this study.

ASH2L-mediated H3K4me3 drives diabetic nephropathy through HIPK2 and Notch1 pathway

Diabetic nephropathy (DN) is one of the complications of diabetes. Long-term hyperglycemia in the kidney results in renal insufficiency, and eventually leads to end-stage renal disease. Epigenetic factor ASH2L has long been identified as a transcriptional activator, and we previously indicated that ASH2L aggravated fibrosis and inflammation in high glucose-induced glomerular mesangial cells, but the pathophysiological relevance and the mechanism of ASH2L-mediated H3K4me3 in DN is not well understood. Here we demonstrated that ASH2L is upregulated in glomeruli isolated from db/db mice. Loss of ASH2L protected glomerular injury caused by hyperglycemia, as evidenced by reduced albuminuria, preserved structure, decreased glomerular extracellular matrix deposition, and lowered renal glomerular expression of proinflammatory and profibrotic markers in db/db mice. Furthermore, we demonstrated that enrichment of ASH2L-mediated H3K4me3 on the promoter regions of ADAM17 and HIPK2 triggered their transcription, leading to aberrant activation of Notch1 signaling pathway, thereby contributing to fibrosis and inflammation in DN. The findings of this study provide compelling evidence for targeting ASH2L as a potential therapeutic strategy to prevent or slow down the progression of DN.

N4BP1 mediates RAM domain-dependent notch signaling turnover during neocortical development.

Notch signaling pathway activity, particularly fluctuations in the biologically active effector fragment NICD, is required for rapid and efficient dynamic regulation of proper fate decisions in stem cells. In this study, we identified NEDD4-binding protein 1 (N4BP1), which is highly expressed in the developing mouse cerebral cortex, as a negative modulator of Notch signaling dynamics in neural progenitor cells. Intriguingly, N4BP1 regulated NICD stability specifically after Notch1 S3 cleavage through ubiquitin-mediated degradation that depended on its RAM domain, not its PEST domain, as had been extensively and previously described. The CoCUN domain in N4BP1, particularly the "Phe-Pro" motif (862/863 amino acid), was indispensable for mediating NICD degradation. The Ring family E3 ligase Trim21 was, in contrast to other NEDD4 family members, required for N4BP1-regulated NICD degradation. Overexpression of N4BP1 in cortical neural progenitors promoted neural stem cell differentiation, whereas neural progenitor cells lacking N4BP1 were sensitized to Notch signaling, resulting in the maintenance of stem-like properties in neural progenitor cells and lower production of cortical neurons.