Expression Of Notch Target Genes Research Articles

A NOTCH2 pathogenic variant and HES1 regulate osteoclastogenesis in induced pluripotent stem cells

Hajdu Cheney Syndrome (HCS), a monogenic disorder associated with NOTCH2 pathogenic variants, presents with neurological, craniofacial and skeletal abnormalities. Mouse models of the disease exhibit osteopenia. To determine the consequences of a HCS pathogenic variant in human cells, induced pluripotent NCRM1 and NCRM5 stem (iPS) cells harboring a NOTCH26949C>T mutation or null for HES1 alleles were created. Parental iPSCs, NOTCH26949C>T, HES1 null and control cells, free of chromosomal aberrations were cultured under conditions of neural crest, mesenchymal and osteogenic cell differentiation, or of embryoid body, hematopoietic and osteoclast cell differentiation. The expected cell phenotype was confirmed by cell surface markers and gene signature. NOTCH26949C>T cells displayed enhanced expression of Notch target genes demonstrating the presence of a NOTCH2 gain-of-function. There was a modest enhancement of osteogenesis in NOTCH26949C>T cells manifested by increased mineralized nodule formation and SP7, ALPL and BGLAP mRNA expression. There was enhanced osteoclastogenesis in NOTCH26949C>T cells as evidenced by increased number of osteoclasts and a transient increase in ACP5, CALCR and CTSK transcripts. Osteoblastogenesis was minimally affected by the HES1 deletion, but osteoclast differentiation was significantly impaired. In conclusion, a NOTCH2 pathogenic variant causes modest increases in osteoblastogenesis and osteoclastogenesis and HES1 is required for osteoclast differentiation in human iPS cells in vitro.

Notch signaling regulates limb regeneration through Hes1 and HeyL in the Chinese mitten crab

Tissue regeneration is an efficient strategy developed by animals to compensate for damaged tissues, involving various types of progenitor cells. Deciphering the signal network that modulates the activity of these progenitors during regeneration is crucial for understanding the differences in regenerative capacities across species. In this study, we evaluated the expression profile and phenotypic function of Notch signaling during limb regeneration in arthropod Chinese mitten crabs. The expression of key components of the Notch signaling pathway was upregulated at 7-day post-autotomy (7 DPA), and declined later at 18-day post-autotomy (18 DPA). To assess the role of Notch, we injected dsRNA targeting the Notch gene into the automized area and evaluated the regeneration efficiency. Our results indicated that blocking Notch signaling led to regenerative defects, manifested by delays in the wound closure and blastema emergence processes. Furthermore, the expression of Notch target genes, Hes1 and HeyL, was significantly reduced following Notch knockdown by dsRNA. Knockdown of Hes1 specifically impaired the proliferation and expression of neural progenitor cell markers, without affecting myogenic cells. In contrast, blockage of HeyL inhibited the proliferation and expression of markers in both activated neurogenic and myogenic progenitor cells, while up-regulating markers of quiescent neural progenitor cells. These findings suggest that Notch signaling plays an important role in limb regeneration of E. sinensis by activating downstream effectors Hes1 and HeyL, regulating neurogenesis and myogenesis through distinct mechanisms.

Notch1 Phase Separation Coupled Percolation facilitates target gene expression and enhancer looping

The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.

Notch1 Phase Separation Coupled Percolation facilitates target gene expression and enhancer looping.

The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.

Abstract 147: Monocyte-derived Extracellular Delta-like 4 Contributes To Pulmonary Hypertension Progression By Inhibiting Endothelial Notch1 Activation

Background: We published that reduced Notch1 cleavage in lung endothelial cells (ECs) contributes to pulmonary hypertension (PH) development. However, the underlying mechanism of Notch1 inactivation remains elusive. We recently found enhanced circulating extracellular Delta-like ligand 4 (exDll4) from monocytes in HIV patients, the population with persistent systemic inflammation. Mobilized Notch ligands have demonstrated the ability to inhibit Notch signaling in both in vitro and in vivo studies. Hence, we hypothesize that inflammation increases monocyte-derived exDll4 and diminishes Notch1 cleavage in lung ECs, subsequently promoting the progression of PH. Methods and Results: Tumor necrosis factor-alpha (TNFα) is a major cytokine increase in the plasma of PAH patients and mouse models. Our in vitro studies revealed that TNFα induced a 17.1-fold and 23.6-fold increase of Dll4 mRNA and protein in human monocytes and a 7.2-fold elevation in exDll4 secretion. In PH mouse models induced by chronic hypoxia, Dll4 protein expression in circulating monocytes increased by 4.2 folds, and plasma exDll4 levels rose by 2.6 folds. In PAH patients, serum exDll4 was elevated by 3 folds compared to non-PAH patients. Mimicking enhanced exDll4 in PH patients; we found that exDll4 injection exacerbated PH progression in hypoxia-treated mice, accompanied by inhibited EC-Notch1 cleavage, leaky EC cell-cell junctions, and increased immune cell infiltration in the lungs. Notably, specific knock-out monocyte Dll4 in mice (mDll4 KO mice) significantly attenuated PH progression, indicated by reduced right ventricular systolic pressure and right ventricular remodeling. Mechanistically, exDll4 treatment significantly inhibited Notch1 activation and Notch target gene expression in human pulmonary microvascular ECs (HPMECs) and led to cell apoptosis, cell arrest, impaired barrier function, and enhanced inflammation. Conclusion: Our study demonstrates the role of monocyte-derived exDll4 in PH progression by inducing EC arrest and apoptosis and impairing barrier function in lung ECs through inhibition of Notch1 signaling. These findings imply that monocyte-derived Dll4 could be a potential therapeutic target and biomarker for PH.

Comprehensive genomic features indicative for Notch responsiveness.

Transcription factor RBPJ is the central component in Notch signal transduction and directly forms a coactivator complex together with the Notch intracellular domain (NICD). While RBPJ protein levels remain constant in most tissues, dynamic expression of Notch target genes varies depending on the given cell-type and the Notch activity state. To elucidate dynamic RBPJ binding genome-wide, we investigated RBPJ occupancy by ChIP-Seq. Surprisingly, only a small set of the total RBPJ sites show a dynamic binding behavior in response to Notch signaling. Compared to static RBPJ sites, dynamic sites differ in regard to their chromatin state, binding strength and enhancer positioning. Dynamic RBPJ sites are predominantly located distal to transcriptional start sites (TSSs), while most static sites are found in promoter-proximal regions. Importantly, gene responsiveness is preferentially associated with dynamic RBPJ binding sites and this static and dynamic binding behavior is repeatedly observed across different cell types and species. Based on the above findings we used a machine-learning algorithm to predict Notch responsiveness with high confidence in different cellular contexts. Our results strongly support the notion that the combination of binding strength and enhancer positioning are indicative of Notch responsiveness.

Detection of native, activated Notch receptors in normal human apocrine-bearing skin and in hidradenitis suppurativa.

Notch signalling has generated considerable interest as a pathogenetic factor and a drug target in a range of human diseases. The gamma-secretase complex is crucial in the activation of Notch receptors by cleaving the intracellular domain allowing nuclear translocation. In recent years several mutations in gamma-secretase components have been discovered in patients with familial hidradenitis suppurativa (HS). This has led to hypotheses that impaired Notch signalling could be an important driver for HS in general, not only in the monogenic variants. However, no study has examined insitu Notch activation per se in HS, and some reports with conflicting results have instead been based on expression of Notch receptors or indirect measures of Notch target gene expression. In this study we established immunostaining protocols to identify native, activated Notch receptors in human skin tissue. The ability to detect changes in Notch activation was confirmed with an exvivo skin organ model in which signal was reduced or obliterated in tissue exposed to a gamma-secretase inhibitor. Using these methods on skin biopsies from healthy volunteers and a general HS cohort we demonstrated for the first time the distribution of active Notch signalling in human apocrine-bearing skin. Quantification of activated NOTCH1 & NOTCH2 revealed similar levels in non-lesional and peri-lesional HS to that of healthy controls, thus ruling out a general defect in Notch activation in HS patients. We did find a variable but significant reduction of activated Notch in epidermis of lesional HS with a distribution that appeared related to the extent of surrounding tissue inflammation.

Synergistic regulation of Notch signaling by different O-glycans promotes hematopoiesis.

Glycosylation of Notch receptors by O-fucose glycans regulates Notch ligand binding and Notch signaling during hematopoiesis. However, roles in hematopoiesis for other O-glycans that modify Notch receptors have not been determined. Here we show that the EGF domain specific GlcNAc transferase EOGT is required in mice for the optimal production of lymphoid and myeloid cells. The phenotype of Eogt null mice was largely cell-autonomous, and Notch target gene expression was reduced in T cell progenitors. Moreover, EOGT supported residual Notch signaling following conditional deletion of Pofut1 in hematopoietic stem cells (HSC). Eogt : Pofut1 double mutant HSC had more severe defects in bone marrow and in T and B cell development in thymus and spleen, compared to deletion of Pofut1 alone. The combined results show that EOGT and O-GlcNAc glycans are required for optimal hematopoiesis and T and B cell development, and that they act synergistically with POFUT1 and O-fucose glycans to promote Notch signaling in lymphoid and myeloid differentiation.

Familial Alzheimer's disease-associated PSEN1 mutations affect neurodevelopment through increased Notch signaling.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, but its root cause may lie in neurodevelopment. PSEN1 mutations cause the majority of familial AD, potentially by disrupting proper Notch signaling, causing early unnoticed cellular changes that affect later AD progression. While rodent models are useful for modeling later stages of AD, human induced pluripotent stem cell-derived cortical spheroids (hCSs) allow access to studying the human cortex at the cellular level over the course of development. Here, we show that the PSEN1 L435F heterozygous mutation affects hCS development, increasing size, increasing progenitors, and decreasing post-mitotic neurons as a result of increased Notch target gene expression during early hCS development. We also show altered Aβ expression and neuronal activity at later hCS stages. These results contrast previous findings, showing how individual PSEN1 mutations may differentially affect neurodevelopment and may give insight into fAD progression to provide earlier time points for more effective treatments.

MYCN Amplification Is Associated with Reduced Expression of Genes Encoding γ-Secretase Complex and NOTCH Signaling Components in Neuroblastoma.

Amplification of the MYCN oncogene is found in ~20% of neuroblastoma (NB) cases and correlates with high-risk disease and poor prognosis. Despite the plethora of studies describing the role of MYCN in NB, the exact molecular mechanisms underlying MYCN's contribution to high-risk disease are not completely understood. Herein, we implemented an integrative approach combining publicly available RNA-Seq and MYCN ChIP-Seq datasets derived from human NB cell lines to define biological processes directly regulated by MYCN in NB. Our approach revealed that MYCN-amplified NB cell lines, when compared to non-MYCN-amplified cell lines, are characterized by reduced expression of genes involved in NOTCH receptor processing, axoneme assembly, and membrane protein proteolysis. More specifically, we found genes encoding members of the γ-secretase complex, which is known for its ability to liberate several intracellular signaling molecules from membrane-bound proteins such as NOTCH receptors, to be down-regulated in MYCN-amplified NB cell lines. Analysis of MYCN ChIP-Seq data revealed an enrichment of MYCN binding at the transcription start sites of genes encoding γ-secretase complex subunits. Notably, using publicly available gene expression data from NB primary tumors, we revealed that the expression of γ-secretase subunits encoding genes and other components of the NOTCH signaling pathway was also reduced in MYCN-amplified tumors and correlated with worse overall survival in NB patients. Genetic or pharmacological depletion of MYCN in NB cell lines induced the expression of γ-secretase genes and NOTCH-target genes. Chemical inhibition of γ-secretase activity dampened the expression of NOTCH-target genes upon MYCN depletion in NB cells. In conclusion, this study defines a set of MYCN-regulated pathways that are specific to MYCN-amplified NB tumors, and it suggests a novel role for MYCN in the suppression of genes of the γ-secretase complex, with an impact on the NOTCH-target gene expression in MYCN-amplified NB.

Abstract 4955: Kurarinone, a natural notch signaling inhibitor and modulator of breast cancer stemness

Abstract Aberrant activation of Notch signaling pathway plays an important role in breast cancer development, progression, and stem cell renewal. Activation of Notch signaling is triggered by the interaction of Notch receptors with different ligands expressed on neighboring cells that bind and induce a second cleavage by a membrane-tethered metalloprotease. The remaining membrane-bound fragment is cleaved by γ-secretase (GS) releasing the Notch Intracellular Domain (NICD), which translocate to the nucleus to activate the expression of Notch target genes. Current indirect approaches to inhibit Notch signaling are based on agents that bind to GS substrates. In the search of novel Notch signaling inhibitor(s), we screened a number of plant flavonoids for their binding potential to GS substrate. Our in-silico study identified kurarinone (KU), a lavandulyl flavanone, having ability to bind to GS complex. The KU-GS complex was structurally and energetically stable during the molecular dynamics simulation period. Interestingly, dose and time dependent toxicity of KU was observed in T47D and MDA-MB-231 breast cancer cells at 1-25µM concentration. KU treatment of cells led to inhibition in cyclin D1 expression, arrest of cells in the G1 phase of the cell cycle and subsequently undergoes apoptosis. KU-mediated apoptosis was a result of altered mitochondria membrane potential and increased ROS production. In dual-luciferase Notch promoter assay, KU inhibited GS-mediated NICD production in transfected HEK293 cells. Furthermore, KU treatment decreased the mammosphere formation potential of MCF-7 cells through altered gene expression involved in self-renewal and stemness. KU treated MDA-MB-231 cells and MCF-7 derived mammosphere showed downregulation of Notch signaling through alteration in the expression of downstream genes such as Hey1, Hes1, and E-cadherin at both transcript and protein levels. Taken together, the present study identified KU as a novel natural Notch signaling inhibitor that could provide a novel and safer approach for the treatment of breast cancer through reduction in stemness and self-renewal properties. Citation Format: Prem Prakash Kushwaha, Mohd Shuaib, Kumari Sunita Prajapati, Atul Kumar Singh, Mahesh Kulharia, Shashank Kumar, Sanjay Gupta. Kurarinone, a natural notch signaling inhibitor and modulator of breast cancer stemness. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4955.

Histone methyltransferase SETD1A interacts with notch and promotes notch transactivation to augment ovarian cancer development

BackgroundHigh expression of SETD1A, a histone methyltransferase that specifically methylates H3K4, acted as a key oncogene in several human cancers. However, the function and underlying molecular mechanism of SETD1A in ovarian cancer (OV) remain markedly unknown.MethodsThe expression of SETD1A in OV were detected by Western blot and analyzed online, and the prognosis of STED1A in OV were analyzed online. The protein and mRNA levels were determined by Western blot and RT-qPCR. The cell proliferatin, migration and invasion were measured by CCK-8 and transwell assays. The protein interaction was detected by co-IP assay. The interaction between protein and DNA was performed by ChIP assay. The tumor growth in vivo was performed by xenograft tumor model.ResultsSETD1A was overexpressed in OV and a predictor of poor prognosis. Overexpression of SETD1A augmented the abilities of cell proliferation, migration, and invasion in MRG1 and OVCAR5 cells. In comparison, SETD1A knockdown suppressed cell growth, migration, and invasion in SKOV3 and Caov3 cells. Specifically, SETD1A enhanced Notch signaling by promoting the expression of Notch target genes, such as Hes1, Hey1, Hey2, and Heyl. Mechanistically, SETD1A interacted with Notch1 and methylated H3K4me3 at Notch1 targets to enhance Notch signaling. In addition, restoration of Notch1 in SETD1A-knockdown OV cells recovered cell proliferation, migration and invasion, which was inhibited by SETD1A knockdown. Furthermore, reduction of SETD1A suppressed tumorigenesis in vivo.ConclusionIn conclusion, our results highlighted the key role of SETD1A in OV development and proved that SETD1A promotes OV development by enhancing Notch1 signaling, indicating that SETD1A may be a novel target for OV treatment.

Dormancy Is a Feature of Therapy Resistant Mouse and Human Leukemia-Initiating Cells in T-ALL

Addressing therapy failure and relapse is critical in improving outcomes for pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Relapse is thought to represent a failure to effectively eliminate leukemia-initiating cells (L-ICs), which survive chemotherapy and self-renew, regenerating the full complement of leukemic cell populations. Results from mouse T-ALL models show that the double negative-3 (DN3) thymic progenitors are enriched for L-IC activity; however, the L-IC remains an undefined subset of this population. We used single cell RNA-sequencing (scRNA-seq) to profile leukemic development in vivo, with a focus on uncovering DN3 heterogeneity. Integrated UMAP clustering of scRNA-seq libraries from wild type (WT), Tal1/Lmo2 preleukemic (PL), and Tal1/Lmo2 T-ALL (Leuk) thymi revealed an expansion of DN3 progenitors in PL and Leuk samples with decreases in mature (single positive) thymocytes. Unlike WT thymocytes, where Notch1 target gene expression was restricted to DN3 cells, Notch1-Myc pathway activation was maintained throughout thymocyte development in Tal1/Lmo2 transgenic mice. Re-clustering of DN3 cells revealed the presence of dormant and proliferative DN3 cells in PL and Leuk thymus. Dormant DN3 signatures resembled quiescent, Notch1-active DN3a (pre-β-selection) thymocytes and proliferative DN3 cells were transcriptionally similar to DN3b thymocytes. Dormant mouse DN3 cells were enriched for signatures of quiescent, therapy resistant ALL patient cells and cells obtained from ALL patients at minimal residual disease (Ebinger et al., Cancer Cell 2016), suggesting that dormant leukemic DN3 cells resemble human L-ICs that mediate drug resistance and relapse. To test this, we employed in vivo nucleosome labeling to profile dormant DN3 cells throughout leukemia development and functionally evaluate their response to chemotherapy. To assess cell divisional history, WT, Tal1, Lmo2, or Tal1/Lmo2 mice harboring the tet-inducible H2B-GFP reporter allele were subjected to in vivo doxycycline pulse (labeling), followed by a two-week chase. Analysis of pulse-chased thymocytes revealed low frequencies of label retaining (GFPHI) DN3 cells in WT mice. However, label retention was significantly increased in Lmo2 DN3 thymocytes (2.9-fold), consistent with other Lmo2 models (Tremblay et al., Nat. Commun. 2018). Tal1 expression also promoted DN3 label retention (6.0-fold) and Tal1/Lmo2 co-expression resulted in further increases in DN3 GFPHI cells (25-fold), indicating that Tal1 and Lmo2 cooperate to promote DN3 dormancy. DN3 GFPHI cells were present at similar frequency in mice with overt T-ALL and in line with scRNA-seq data, GFPHI cells were observed nearly exclusively as a subset of the leukemic DN3a population. Treatment with Vincristine, DeXamethasone, and L-asparaginase (VXL) led to a further increase in GFPHI DN3 frequency in Lmo2 and Tal1 preleukemic mice (3.1- and 9.7-fold, respectively), suggesting that cell cycle restricted L-ICs may evade conventional chemotherapy and mediate relapse. We then tested whether dormancy is a feature of the L-IC in T-ALL patients. Analysis of relapsed pediatric T-ALL cells revealed that the CD7+CD1a- L-IC population (Chiu et al., Blood 2010) is cell cycle restricted compared to CD7+CD1a+ leukemic cells and that VXL treatment enriched for CD7+CD1a- cells in xenografted mice, suggesting that cell cycle restriction may be an important feature of both mouse and human T-ALL L-ICs. Analysis of transcriptomes to reveal mediators of dormancy that may be exploited to chemosensitize L-ICs will be discussed.

Rutin Potentially Binds the Gamma Secretase Catalytic Site, Down Regulates the Notch Signaling Pathway and Reduces Sphere Formation in Colonospheres.

Rutin, a natural flavonol, can modulate molecular signaling pathways and has considerable potential in cancer treatment. However, little is known about the effect of rutin on the notch signaling pathway (NSP) in cancer and cancer stem-like cells. In this study, we explored the effect of rutin on gamma secretase (GS, a putative notch signaling target) inhibition mediated NICD (Notch Intracellular Domain) production in colon cancer cells. Molecular docking, MM-GBSA, and Molecular dynamics (MD) simulation experiments were performed to check rutin’s GS catalytic site binding potential. The HCT-116 colon cancer and cancer stem-like cells (colonospheres) were utilized to validate the in silico findings. The NICD production, notch promoter assay, expression of notch target genes, and cancer stemness/self-renewal markers were studied at molecular levels. The results were compared with the Notch-1 siRNA transfected test cells. The in silico study revealed GS catalytic site binding potential in rutin. The in vitro results showed a decreased NICD formation, an altered notch target gene (E-cad, Hes-1, and Hey-1) expression, and a reduction in stemness/self-renewal markers (CD44, c-Myc, Nanog, and Sox2) in test cells in a time and dose-dependent manner. In conclusion, rutin inhibits the notch signaling pathway and reduces the stemness/self-renewal property in colon cancer cells and the colonospheres by targeting gamma secretase. The clinical efficacy of rutin in combination therapy in colon cancer may be studied in the future.

Abstract 204: Notch2NL is a novel regulator of radiation sensitivity in non-small cell lung cancer and a positive regulator of MYC activity

Abstract Background: A gain-of-function genetic screen identified NOTCH2 N-terminal like (NOTCH2NL) as a candidate novel gene involved in radiation resistance in non-small cell lung cancer (NSCLC). NOTCH2NL is located on chromosome position 1q21.1 which was incorrectly aligned in previous builds of the reference human genome sequence. NOTCH2NL was derived from a segmental duplication event in NOTCH2 during the evolutionary divergence between humans and non-human primates, forming three Notch2NL proteins: Notch2NL-A, Notch2NL-B and Notch2NL-C. Consequently, the only species whose genomes contain NOTCH2NL are modern and ancient humans. Notch2NL-B is a secreted protein and has been shown to promote Notch signalling in cortical stem cell progenitors via inhibition of inhibitory cis-DLL1/NOTCH signalling. Methods: Stable overexpression of NOTCH2NLB and secreted Notch2NL-B protein were validated by RT-qPCR and Western Blotting. The effects of NOTCH2NLB overexpression; transfer of Notch2NL-B protein from conditioned media onto wild-type cell lines; and siRNA knockdown of NOTCH1-4 transmembrane receptors on Notch signalling were quantified by a Notch luciferase reporter assay, RT-qPCR of classical NOTCH target genes and cancer stem cell marker (CSC) and RNA-Seq. Clonogenic cell survival assays determined the impact of NOTCH2NLB overexpression on radiation response. Cell proliferation was measured using the IncuCyte Zoom system. shRNA stable cell lines to knockdown NOTCH2NL were generated to mitigate the NOTCH2NLB overexpression phenotype we observed. RNA-Seq data from the first 100 patients from the TRACERx lung cancer study was used to quantify hallmark gene expression signatures and NOTCH2NLB expression. Results: NOTCH2NLB overexpression upregulates CSL-dependent Notch reporter luciferase activity; confers a radiation-resistant phenotype and impacts proliferation rate in NSCLC cell lines. Furthermore, we demonstrate that NOTCH2NLB overexpression and secreted Notch2NL-B protein transfer increase expression of classical NOTCH target genes (c-MYC, HES1 and HEY1) and CSC marker, ALDH1A1. Analysis of RNA-Seq data from the Lung TRACERx cohort study shows that NOTCH2NL is commonly expressed in NSCLC. NOTCH2NL expression positively correlates with notch hallmark gene expression signature in squamous (R=0.59, p=5.6e-9) and non-squamous carcinoma (R=0.51, p=4.7e-10). Interestingly, NOTCH2NL expression also positively correlated with the myc hallmark gene expression signature in squamous cell carcinoma (R=0.45, p=2.4e-5). Conclusions: Notch2NL is a novel determinant of radiation therapy response and impacts canonical Notch signalling in lung cancer. It is commonly expressed in NSCLC and can act in a paracrine manner as a secreted protein. Notch2NL, through canonical notch signalling, can regulate MYC expression and increase activity in the MYC pathway. Citation Format: Simranpreet Kaur Summan, Barbara Chow, Rhona Millar, Su Kit Chew, Philip East, Robert Hynds, Carlos Martinez Ruiz, Eva Gronroos, Mariam Jamal-Hanjani, Kevin Litchfield, Nicholas McGranahan, Nnennaya Kanu, Charles Swanton, Crispin Thomas Hiley. Notch2NL is a novel regulator of radiation sensitivity in non-small cell lung cancer and a positive regulator of MYC activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 204.

Abstract 5672: Notch3 signaling between myeloma cells and osteocytes in the tumor niche promotes tumor growth and bone destruction

Abstract In multiple myeloma (MM), Notch signaling, a pathway mediating cell-to-cell communication between cells in the tumor niche, promotes MM proliferation and bone destruction. We previously reported that osteocytes (Ots), the most abundant bone cells, provide a microenvironment conducive for MM progression and bone destruction. Ots activate Notch signaling, increase Notch3 expression, and stimulate proliferation in MM cells. We detected Notch3 in CD138+ cells from MM patients and human and murine MM cell lines. Yet, the role of Notch3 in MM is unknown. Here, we used a shRNA approach to knockdown Notch3 to study its role in MM cells and their communication with Ots. The protein levels of active Notch3 (NICD3) were decreased by 80% in 5TGM1 MM cells transduced with shRNA against Notch3 compared to control shRNA-control cells. In contrast, Notch1, 2, and 4 NICD protein levels remained unchanged compared to controls. Notch3 knockdown decreased Notch target gene and cyclinD1 expression, reduced proliferation by 35%, and modestly increased apoptosis in MM cells. Additionally, Notch3 knockdown decreased Rankl expression and the ability of MM cells to promote osteoclastogenesis in vitro. Consistent with these observations, bioinformatic analysis of the transcriptome of CD138+ cells from newly diagnosed patients revealed upregulated processes related to positive regulation of cell proliferation and osteoclastogenesis in MM patients with high vs. low Notch3 expression. Next, we examined the role of Notch3 in MM-Ots communication. Notch3 knockdown in MM cells partially prevented the upregulation of Notch target genes and cyclinD1 expression and proliferation induced by direct contact with Ots. Inhibition of all Notch receptors with GSI fully prevented osteocyte-induced proliferation and Notch activation, suggesting that in addition to Notch3, other Notch receptors mediate MM-osteocyte communication. Remarkably, shRNA-mediated Notch2 inhibition did not alter MM cell proliferation or communication with osteocytes. Lastly, we analyzed the effects of Notch3 knockdown in MM cells in ex vivo and in vivo models. Using ex vivo bone organ cultures, we found less MM proliferation and lower levels of the resorption marker CTX in conditioned media from bones cultured with shRNA-Notch3 MM cells compared to control bones cultured alone. We injected mice intratibially with shRNA-Notch3 or shRNA-control 5TGM1 MM cells. After 5 weeks, mice bearing shRNA-Notch3 cells had a 50% decrease in tumor burden, 50% reduction in osteolytic lesions, and exhibited 30% more cancellous bone compared to mice bearing control MM cells. Together, these preclinical and clinical findings support that Notch3 signaling is a crucial mediator of homotypic and heterotypic communication in the MM tumor niche. Future studies are needed to evaluate Notch3 in the tumor microenvironment as a therapeutic target for the treatment of MM. Citation Format: Hayley M. Sabol, Tania Amorim, Cody Ashby, David Halladay, Judith Anderson, Meloney Cregor, Megan Sweet, Intawat Nookaew, Noriyoshi Kurihara, G. David Roodman, Teresita Bellido, Jesus Delgado-Calle. Notch3 signaling between myeloma cells and osteocytes in the tumor niche promotes tumor growth and bone destruction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5672.

Non-classical Notch signaling by MDA-MB-231 breast cancer cell-derived small extracellular vesicles promotes malignancy in poorly invasive MCF-7 cells.

Aberrant Notch signaling is implicated in breast cancer progression, and recent studies have demonstrated links between the Notch pathway components Notch1 and Notch1 intracellular domain (N1ICD) with poor clinical outcomes. Growing evidence suggests that Notch signaling can be regulated by small extracellular vesicles (SEVs). Here, we used breast cancer cell models to examine whether SEVs are involved in functional Notch signaling. We found that Notch components are packaged into MDA-MB-231- and MCF-7-derived SEVs, although higher levels of N1ICD were detected in SEVs from the more aggressive MDA-MB-231 cell line than from poorly invasive MCF-7 cells. SEV-Notch components were functional, as SEVs cargo from MDA-MB-231 cells induced the expression of Notch target genes in MCF-7 cells and triggered a more invasive and proliferative phenotype concomitant with the acquisition of mesenchymal features. Neutralization of the N1ICD cargo in MDA-MB-231-derived SEVs significantly reduced their potential to enhance the aggressiveness of MCF-7 cells in vitro and in a xenograft model. Overall, our results indicate that a SEV-mediated non-classical pathway of Notch signal transduction in breast cancer models bypasses the need for classical ligand-receptor interactions, which may have important implications in cancer.

NOTCH4 Exhibits Anti-Inflammatory Activity in Activated Macrophages by Interfering With Interferon-γ and TLR4 Signaling.

NOTCH4 is a member of the NOTCH family of receptors whose expression is intensively induced in macrophages after their activation by Toll-like receptors (TLR) and/or interferon-γ (IFN-γ). In this work, we show that this receptor acts as a negative regulator of macrophage activation by diminishing the expression of proinflammatory cytokines, such as IL-6 and IL-12, and costimulatory proteins, such as CD80 and CD86. We have observed that NOTCH4 inhibits IFN-γ signaling by interfering with STAT1-dependent transcription. Our results show that NOTCH4 reprograms the macrophage response to IFN-γ by favoring STAT3 versus STAT1 phosphorylation without affecting their expression levels. This lower activation of STAT1 results in diminished transcriptional activity and expression of STAT1-dependent genes, including IRF1, SOCS1 and CXCL10. In macrophages, NOTCH4 inhibits the canonical NOTCH signaling pathway induced by LPS; however, it can reverse the inhibition exerted by IFN-γ on NOTCH signaling, favoring the expression of NOTCH-target genes, such as Hes1. Indeed, HES1 seems to mediate, at least in part, the enhancement of STAT3 activation by NOTCH4. NOTCH4 also affects TLR signaling by interfering with NF-κB transcriptional activity. This effect could be mediated by the diminished activation of STAT1. These results provide new insights into the mechanisms by which NOTCH, TLR and IFN-γ signal pathways are integrated to modulate macrophage-specific effector functions and reveal NOTCH4 acting as a new regulatory element in the control of macrophage activation that could be used as a target for the treatment of pathologies caused by an excess of inflammation.

Regulation of Notch Signaling By O-Glycans during Lymphopoiesis and Myelopoiesis

IntroductionNotch signaling is essential for the optimal generation of T, B and myeloid cells. Epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch receptors are modified by O-fucose and O-GlcNAc glycans transferred by protein O-fucosyltransferase 1 (POFUT1) and EGF O-GlcNAc-transferase (EOGT), respectively. EOGT promotes Notch ligand binding and Notch signaling in the developing retina in mice, and its loss is the basis of Adam's Oliver Syndrome 4 (AOS4) in humans. The loss of POFUT1 in humans leads to the Dowling Degos Disease 2 (DDD2). Thus, O-fucose and O-GlcNAc glycans may have different functions in Notch signaling, reflected in different requirements for T, B or myeloid cell development.Methods:Eogt control, Eogt null, Pofut1 floxed and Vav1-iCre transgenic mice were used to generate single and compound mutant mice with inactive Eogt, Pofut1 or both Eogt and Pofut1. Antibody markers of hematopoietic stem progenitor cell (HSPC), lymphoid and myeloid subsets were used to identify different T, B and myeloid cell subsets by flow cytometry using the Cytek TM Aurora Flow Cytometer. FCS files were analyzed using FlowJo software (BD). CD45.1+ (B6.SJL-Ptprc a Pepc b/BoyJ #002014) congenic mice were used for bone marrow transfer. Expression of NOTCH1 and binding of soluble Notch ligands to DN T cells was analyzed by flow cytometry. Expression of different Notch target genes was determined using qRT-PCR anlaysis.Results:Eogt null mice exhibited altered production of certain T cell subsets in thymus and B cell subsets in spleen, most similar to alterations observed in mice lacking LFNG, MFNG and RFNG. This phenotype was cell-autonomous as Eogt null bone marrow cells transferred defective T and B cell development to irradiated recipient mice. More severe defects in hematopoiesis were observed in mice conditionally lacking Pofut1 in hematopoietic stem cells (HSC) via Vav1-iCre. However , deletion of both O-fucose and O-GlcNAc glycans together in Eogt:Pofut1 double knockout HSC, led to the most severe decrease in common lymphoid and myeloid progenitors in bone marrow. In thymus, the dramatic reduction in T cell subsets observed in Pofut1 cKO mice was reduced further (in DN2 subsets) in Eogt:Pofut1 dKO mice. In spleen, there was a significantly greater decrease in follicular B and other B cell populations, and a greater increase in CD11b/c+ and Gr1+ myeloid cells in Eogt:Pofut1 dKO mice. Splenomegaly occurred in both Pofut1 cKO and Eogt:Pofut1 dKO mice, with a greater increase in extramedullary hematopoiesis in the dKO spleen. Binding of soluble DLL4-Fc was >90% reduced in Pofut1 cKO DN T cells, and not further reduced in dKO cells, while NOTCH1 expression at the cell surface was only slightly reduced. The relative expression of Notch target genes including Hes1, CD25, cMyc, and Deltex1 was similarly reduced in Pofut1 cKO and Eogt:Pofut1 dKO DN T cell progenitors.Conclsuion:The combined data suggest that O-fucose and O-GlcNAc glycans act in an additive fashion to optimize Notch signaling in lymphoid and myeloid differentiation.Keywords: Notch Signaling, O-Glycans, Protein O-fucosyltransferase 1 (POFUT1), O-GlcNAc transferase (EOGT), Lymphopoiesis, Myelopoiesis. DisclosuresStanley: Aviceda Therapeutics, Inc.: Consultancy, Current holder of stock options in a privately-held company.

Development of Notch1 Positive T-Lineage Lymphomas or Splenic Marginal Zone Lymphomas with Pan-Hematopoietic or Pro-B Cell Specific Deletion of Trp53 with Distinct Differentially Dysregulated Pathways

Deletion of the tumor suppressor gene TP53 (Trp53 in mice) has been associated with the development of numerous human malignancies. TP53 acts as a central coordinator of the DNA damage response. In mice, pan-Trp53 deletion leads predominantly to the development of T-cell lymphomas, followed by B-cell lymphomas, sarcomas and teratomas. In order to dissect the role of Trp53 in the hematopoietic system, we created two different loss of function mouse models: Pan-hematopoietic Trp53 deletion using Vav1-Cre based deletion; and a B-cell-specific deletion was created using CD19-Cre.Vav1-p53CKO mice developed hematolymphoid malignancies with 100% penetrance by 12 months. Most malignancies observed were CD3e+ T-lineage lymphomas involving the thymus or spleen (37/45). Beyond 200 days, these mice predominantly developed mixed myeloid malignancies. The shift away from T-lineage malignancies in older mice may reflect aging-related decline of pre-malignant lymphoid progenitors and skewing to myeloid progenitors.Flow cytometric characterization of the T-lineage lymphomas identified a mix of tumors, including double-negative (CD4-CD8-), double-positive (CD4+CD8+), or single positive (CD4/CD8). In pre-malignant mice, Vav1-p53CKO thymocytes showed accelerated maturation with most of the cells in the DN4 stage, suggesting a bypass of the p53-dependent DN3 β-selection checkpoint. All T-lineage lymphomas showed overexpression of surface Notch1 as well as overexpression of Notch1 targets Hes1 and p21 at the transcript level. Consistent with prior data, normal murine thymocyte subsets showed high levels of expression of Notch1 target genes at the DN3 stage of development, which appears dysregulated in these T-lineage lymphomas. This Notch1 activation was found to be multifactorial with increased Mdm2 and decreased Numb levels seen in tumors. Overall, we demonstrate Notch1 activation and subsequent acceleration through the T-cell developmental stages in this model of pan-hematopoietic Trp53 deletion.The B-cell specific Trp53 knockout mice (CD19-P53CKO) (n=54) were followed up for up to 2 years. The majority (47/54) developed splenomegaly in an age-dependent manner. Histologic examination showed marginal zone expansion (6/54), frank low-grade marginal zone lymphoma (16/54) or diffuse splenic lymphoma (25/54). The disease was confined to the spleen in the case of lower-grade histology while higher grades correlated with liver and kidney involvement. Flow cytometric analysis of tumors showed B220+ CD19+ IgM+ cells. Interestingly, these tumors demonstrated low levels of Notch2 expression, which normally is highly expressed in marginal zone B-cells.In order to characterize pathogenesis, we sorted follicular and marginal zone B-cells from floxed P53 and pre-malignant CD19-P53CKO mice. RNA was isolated from all these fractions and the spleens of 5 CD19-P53KO mice with diffuse lymphoma and subjected to RNA-Seq. A comparison of the floxed p53 with the CD19-P53CKO fractions (follicular and marginal zone) revealed a highly similar transcriptome. On the other hand, p53-deficient lymphomas showed >10,000 genes significantly differentially expressed demonstrating the unique transcriptome which developed during malignant transformation.Pathway analysis of these genes using Gene Set Enrichment Analysis (GSEA) identified enrichment of PI3K, Rap1 and MAPK signaling pathways, which are associated with cellular proliferation. Overexpression of the PI3K pathway genes Ccne1, Sgk1, Mapk13 and Pik3cb were validated by qPCR in 10 independent tumor samples when compared to the splenic marginal zone fractions. In the B-cell lineage, Trp53 deficiency leads to the dysregulation of multiple genes involved in key cellular signaling pathways, including the PI3K/MAPK pathway.In summary, pan hematopoietic deletion of Trp53 led to T-lineage lymphoma in young mice and myeloid tumors in older mice; with activation of Notch1 signaling in the former. B-cell specific deletion of Trp53 led to splenic marginal zone and diffuse B-cell lymphoma with transcriptional dysregulation of key signaling molecules. Hence, tumorigenesis by Trp53 deletion is tightly linked to lineage and appears to dysregulate key signaling pathways that are operant in those lineages, potentially identifying novel strategies for therapeutic interventions in P53 dependent human hematolymphoid malignancies. DisclosuresNo relevant conflicts of interest to declare.