Notch Transcriptional Complex Research Articles

Modular Domains within a Super Enhancer Determine Drug Resistance in Leukemia

▪Notch plays roles in T cell development and is a common oncogenic driver in T cell acute lymphoblastic leukemia. Myc is a critical target of Notch in normal and malignant pre-T cells. We and others identified a distal enhancer located >1 Mb 3' of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter (1, 2). Deletion of a 1 kb region surrounding this enhancer resulted in a block in T cell development and inhibited Notch induced T-ALL induction and maintenance (2). Nevertheless, this 1 kb region contains binding sites for multiple transcription factors; thus, the requirement for specific transcription factors has not been resolved. To determine the precise sequences required for function of the distal enhancer, we used CRISPR/CAS9 to specifically mutate transcription factor binding sites in the Notch-dependent Myc enhancer (NDME). Specific mutation of the RBPj binding site blocked Notch/RBPj binding and inhibited Myc expression and T-ALL cell growth/survival. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Specifically mutating the single RBPj binding site abrogated H3K27Ac across this entire 600 kb region. Thus, Notch binding at a single site in the NDME is required for enhancer function and H3K27Ac across a very broad region. Furthermore, the Notch-regulated distal enhancer is comprised of individual enhancers that are uniquely sensitive to Notch or BET domain inhibitors. By selecting tumor cells resistant to each inhibitor, we show that the change in drug sensitivity results from selective use of these individual enhancers that are located within the broad Notch regulated region. These data not only provide insights into enhancer function but suggest strategies to treat T-ALL, including drug resistance, by therapeutically targeting these enhancers.

P53 Modulates Notch Signaling in MCF-7 Breast Cancer Cells by Associating With the Notch Transcriptional Complex Via MAML1.

p53 and Notch-1 play important roles in breast cancer biology. Notch-1 inhibits p53 activity in cervical and breast cancer cells. Conversely, p53 inhibits Notch activity in T-cells but stimulates it in human keratinocytes. Notch co-activator MAML1 binds p53 and functions as a p53 co-activator. We studied the regulation of Notch signaling by p53 in MCF-7 cells and normal human mammary epithelial cells (HMEC). Results show that overexpression of p53 or activation of endogenous p53 with Nutlin-3 inhibits Notch-dependent transcriptional activity and Notch target expression in a dose-dependent manner. This effect could be partially rescued by transfection of MAML1 but not p300. Standard and quantitative co-immunoprecipitation experiments readily detected a complex containing p53 and Notch-1 in MCF-7 cells. Formation of this complex was inhibited by dominant negative MAML1 (DN-MAML1) and stimulated by wild-type MAML1. Standard and quantitative far-Western experiments showed a complex including p53, Notch-1, and MAML1. Chromatin immunoprecipitation (ChIP) experiments showed that p53 can associate with Notch-dependent HEY1 promoter and this association is inhibited by DN-MAML1 and stimulated by wild-type MAML1. Our data support a model in which p53 associates with the Notch transcriptional complex (NTC) in a MAML1-dependent fashion, most likely through a p53-MAML1 interaction. In our cellular models, the effect of this association is to inhibit Notch-dependent transcription. Our data suggest that p53-null breast cancers may lack this Notch-modulatory mechanism, and that therapeutic strategies that activate wild-type p53 can indirectly cause inhibition of Notch transcriptional activity.

Abstract 3938: MafB and ETS2 potentiate Notch signaling in T-ALL

Abstract Despite the high frequency of activating Notch mutations in human T-cell acute lymphoblastic leukemia (T-ALL), these naturally occurring Notch1 mutant alleles fail to efficiently drive leukemia onset, indicating a requirement for collaborating events that potentiate the Notch oncogenic program. We performed an in vitro gain-of-function screen to identify gene products that enhanced the signaling strength of naturally occurring Notch mutants. Both MafB and ETS2 emerged as candidates that individually potentiated Notch signaling, and when co-expressed, they dramatically enhanced signaling strength comparable to the levels induced by MAML1, a core component of the Notch transcriptional complex. Knockdown of MafB in mouse T-ALL cell lines suppressed the expression of common Notch target genes, including cMyc and Hes1, and sustained knockdown of MafB expression impaired T-ALL cell growth in cell competition assays. Notably, MafB is sufficient to enhance the oncogenic potential of naturally occurring Notch mutants in vivo, by significantly accelerating T-ALL onset. Moreover, this enhanced Notch pathway activation required MafB-ETS2 interaction, and facilitated the recruitment of PCAF and p300. Although all primary human T-ALL samples that express MafB display an elevated Notch pathway signature, the T-ALL samples positive for high levels of MafB expression, exhibit low levels of Notch1 expression, further suggesting a role for MafB as an enhancer. These data present a novel mechanism for the enhancement of the oncogenic potential of weak Notch mutants in leukemia and introduce MafB as a potential therapeutic target in T-ALL. Note: This abstract was not presented at the meeting. Citation Format: Kostandin Pajcini, Lanwei Xu, Curtis Lee, Sara Cherry, Warren Pear. MafB and ETS2 potentiate Notch signaling in T-ALL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3938. doi:10.1158/1538-7445.AM2015-3938

Downregulation of RND3/RhoE in glioblastoma patients promotes tumorigenesis through augmentation of notch transcriptional complex activity.

Activation of Notch signaling contributes to glioblastoma multiform (GBM) tumorigenesis. However, the molecular mechanism that promotes the Notch signaling augmentation during GBM genesis remains largely unknown. Identification of new factors that regulate Notch signaling is critical for tumor treatment. The expression levels of RND3 and its clinical implication were analyzed in GBM patients. Identification of RND3 as a novel factor in GBM genesis was demonstrated in vitro by cell experiments and in vivo by a GBM xenograft model. We found that RND3 expression was significantly decreased in human glioblastoma. The levels of RND3 expression were inversely correlated with Notch activity, tumor size, and tumor cell proliferation, and positively correlated with patient survival time. We demonstrated that RND3 functioned as an endogenous repressor of the Notch transcriptional complex. RND3 physically interacted with NICD, CSL, and MAML1, the Notch transcriptional complex factors, promoted NICD ubiquitination, and facilitated the degradation of these cofactor proteins. We further revealed that RND3 facilitated the binding of NICD to FBW7, a ubiquitin ligase, and consequently enhanced NICD protein degradation. Therefore, Notch transcriptional activity was inhibited. Forced expression of RND3 repressed Notch signaling, which led to the inhibition of glioblastoma cell proliferation in vitro and tumor growth in the xenograft mice in vivo. Downregulation of RND3, however, enhanced Notch signaling activity, and subsequently promoted glioma cell proliferation. Inhibition of Notch activity abolished RND3 deficiency-mediated GBM cell proliferation. We conclude that downregulation of RND3 is responsible for the enhancement of Notch activity that promotes glioblastoma genesis.

The role of Notch receptors in transcriptional regulation.

Notch signaling has pleiotropic context-specific functions that have essential roles in many processes, including embryonic development and maintenance and homeostasis of adult tissues. Aberrant Notch signaling (both hyper- and hypoactive) is implicated in a number of human developmental disorders and many cancers. Notch receptor signaling is mediated by tightly regulated proteolytic cleavages that lead to the assembly of a nuclear Notch transcription complex, which drives the expression of downstream target genes and thereby executes Notch's functions. Thus, understanding regulation of gene expression by Notch is central to deciphering how Notch carries out its many activities. Here, we summarize the recent findings pertaining to the complex interplay between the Notch transcriptional complex and interacting factors involved in transcriptional regulation, including co-activators, cooperating transcription factors, and chromatin regulators, and discuss emerging data pertaining to the role of Notch-regulated noncoding RNAs in transcription.

Alternative Super-Enhancer States Determine MYC Sensitivity to Notch and Brd4 Inhibitors in T Lymphoblastic Leukemia/Lymphoma

Gain-of-function NOTCH1 mutations are oncogenic drivers in a high fraction of T-cell lymphoblastic leukemia/lymphoma (T-LL). These mutations variously cause increased production or stabilization of the free intracellular domain of NOTCH1, which regulates gene expression by forming a transcription complex with the DNA-binding factor RBPJ and coactivators of the MAML family. Using expression profiling and ChIP-seq, we have shown that NOTCH1/RBPJ complexes activate most target genes by binding to super-enhancers, large regulatory elements that switch on transcription through long-range interactions with gene promoters. MYC is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates MYC is unknown. To understand which regulatory element(s) regulate MYC expression, we used chromatin conformation capture (3C) assays to test the interaction between putative enhancer(s) and the MYC promoter in T-LL cell lines, and reporter gene assays to confirm enhancer function of candidate sites. We identified a distal site located >1 Mb 3’ of human and murine MYC termed the Notch-dependent MYC enhancer (NDME) that binds Notch transcription complexes and physically interacts with the MYC proximal promoter. An ~1 kb DNA fragment containing this site activates a luciferase reporter gene in a Notch-dependent fashion in T-LL cells but not in heterologous cell types. The Notch binding site lies within a large enhancer region (>600 kb in breadth) containing multiple discrete H3K27ac peaks. Remarkably, acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer region and the MYC promoter that correlate with NOTCH1/RBPJ complex binding and MYC expression. T-LL cells selected for resistance to gamma-secretase inhibitors (GSIs) exhibit epigenetic silencing of the NDME and loss of NDME looping interactions with the MYC promoter, yet maintain MYC expression. 3C analysis of GSI resistant cells shows preferential interaction between the MYC promoter and a more 3’ enhancer element recently described as a BRD4-dependent regulator of MYC expression in acute myeloid leukemia cells. In line with this observation, BRD4 antagonists are potent inhibitors of MYC expression in GSI resistant T-LL cells but not GSI-sensitive cells. We also studied a case of Notch-mutated early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL). ChIP-Seq analysis of the leukemic blasts revealed an “AML-like” MYC enhancer chromatin state, and as predicted from our analysis of cell lines, the blasts rapidly down-regulated MYC in response to BRD4 inhibitor but not in response to GSI. These findings suggest that specific MYC chromatin states predict responsiveness to Notch and BRD4 inhibitors, and provide a rationale for use of Notch and BRD4 inhibitor combinations in Notch-mutated leukemias. DisclosuresNo relevant conflicts of interest to declare.

NACK Is an Integral Component of the Notch Transcriptional Activation Complex and Is Critical for Development and Tumorigenesis

The Notch signaling pathway governs many distinct cellular processes by regulating transcriptional programs. The transcriptional response initiated by Notch is highly cell context dependent, indicating that multiple factors influence Notch target gene selection and activity. However, the mechanism by which Notch drives target gene transcription is not well understood. Herein, we identify and characterize a novel Notch-interacting protein, Notch activation complex kinase (NACK), which acts as a Notch transcriptional coactivator. We show that NACK associates with the Notch transcriptional activation complex on DNA, mediates Notch transcriptional activity, and is required for Notch-mediated tumorigenesis. We demonstrate that Notch1 and NACK are coexpressed during mouse development and that homozygous loss of NACK is embryonic lethal. Finally, we show that NACK is also a Notch target gene, establishing a feed-forward loop. Thus, our data indicate that NACK is a key component of the Notch transcriptional complex and is an essential regulator of Notch-mediated tumorigenesis and development.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

The Molecular Basis of Notch Signaling Regulation: A Complex Simplicity

The Notch receptors have attracted considerable attention for their ability to control cellular functions that regulate embryo development and tissue homeostasis. Notch receptors act by controlling the expression of a specific set of target genes. If Notch signaling system can be so simple, and yet so complex in its pleiotropic effects, then a sophisticated network of regulatory mechanisms is required to maintain the control over the initiation, activity and termination of this signaling pathway. A multitude of regulatory mechanisms has been discovered that controls the interaction of Notch receptors with their ligands, the assembling of a Notch transcriptional activation complex and the termination of Notch signals. The intracellular and extracellular domains of the Notch receptors are synthesized as single proteins, pairing with each other during their trafficking through the exocytotic route. The mechanisms operating in the phase preceding the generation of the heterodimeric signal-competent Notch receptors can be as elaborate and physiologically important as those operating downstream of Notch receptor activation. These regulatory mechanisms, which are essential to understand the role of Notch signaling in human physiology and pathology are reviewed here.

The NOTCH signaling pathway: role in the pathogenesis of T-cell acute lymphoblastic leukemia and implication for therapy

T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is characterized by aberrant activation of NOTCH1 in over 60% of T-ALL cases. The high prevalence of activating NOTCH1 mutations highlights the critical role of NOTCH signaling in the pathogenesis of this disease and has prompted the development of therapeutic approaches targeting the NOTCH signaling pathway. Small molecule gamma secretase inhibitors (GSIs) can effectively inhibit oncogenic NOTCH1 and are in clinical testing for the treatment of T-ALL. Treatment with GSIs and glucocorticoids are strongly synergistic and may overcome the gastrointestinal toxicity associated with systemic inhibition of the NOTCH pathway. In addition, emerging new anti-NOTCH1 therapies include selective inhibition of NOTCH1 with anti-NOTCH1 antibodies and stapled peptides targeting the NOTCH transcriptional complex in the nucleus.

Characterization of CSL (CBF-1, Su(H), Lag-1) Mutants Reveals Differences in Signaling Mediated by Notch1 and Notch2

Notch is a conserved signaling pathway that plays essential roles during embryonic development and postnatally in adult tissues; misregulated signaling results in human disease. Notch receptor-ligand interactions trigger cleavage of the Notch receptor and release of its intracellular domain (NICD) from the membrane. NICD localizes to the nucleus where it forms a transcriptionally active complex with the DNA-binding protein CSL and the coactivator Mastermind (MAM) to up-regulate transcription from Notch target genes. Previous studies have determined the structure of the CSL-NICD-MAM ternary complex and characterized mutations that affect complex assembly in functional assays. However, as CSL is expressed in all cell types, these studies have been limited to analyzing mutations in NICD and MAM. Here, we describe a novel set of cellular reagents to characterize how mutations in CSL affect its function as a transcriptional activator. Using retrovirally transduced embryonic fibroblasts from a CSL-null mouse, we generated cell lines that express either wild-type or mutant CSL molecules. We then analyzed these mutants for defects in Notch1- (NICD1) or Notch2 (NICD2)-mediated activation from two different transcriptional reporters (HES-1 or 4×CBS). Our results show that mutations targeted to the different domains of CSL display significant differences in their ability to adversely affect transcription from the two reporters. Additionally, a subset of CSL mutants is sensitive to whether NICD1 or NICD2 was used to activate the reporter. Taken together, these studies provide important molecular insights into how Notch transcription complexes assemble at different target genes and promoter arrangements in vivo.

463P - Development of Cancer Stem Cells Therapeutics

ABSTRACT Disclosure A.A. Epenetos: Receive directors fees from Trojantec ltd. All other authors have declared no conflicts of interest. Cancer Stem Cells, (CSCs), are responsible for the initiation, metastasis and recurrence of a variety of cancers and may be a key reason for the failure of current therapies. Cancer Stem cells operate through similar signaling pathways as normal stem cells such as Notch, Hedgehog,Wnt and others. Notch signaling is abnormal in many, if not all cancers as shown in cancer stem cell development. Furthermore, it may be that some cancers are ‘addicted’ to Notch signaling. This presents opportunities for tumour-specific intervention. As all the signaling cascades converge on the Notch transcriptional complex (NTC) in the nucleus, we have developed a novel synthetic protein acting as a Notch inhibitor operating at nuclear level. This protein is based on the cell penetrating protein (CPP) antennapedia (Antp) produced together with a dominant-negative truncated mastermind-like protein (DN-MAML) called TR4. TR4 has been shown in vitro and in vivo to have anti-Notch and anti-cancer activity with similar potency to many of the gamma-secretase inhibitors or monoclonal antibodies being developed by others against this pathway, but it is completely specific for the NTC. In summary, a specific Notch inhibitor has shown anticancer activity and could now be considered for clinical development.

Transcriptional inhibition of Notch signaling using cell penetrating fusion proteins.

e13525 Background: Notch signalling is deregulated in a number of cancers and is possibly involved in cancer stem cell development. It is postulated that cancers such as breast can become addicted to Notch signalling presenting opportunities for tumour-specific intervention. Due to the presence of at least 4 cell surface receptors that mediate Notch signalling, conventional targeting agents such as monoclonal antibodies cannot inhibit them all. As all the signalling cascades converge on the Notch transcriptional complex (NTC) in the nucleus, inhibition of gene expression by disrupting this machinery is an attractive strategy to overcome this redundancy. Methods: Towards this, we have developed a fusion protein based on the potent cell penetrating protein (CPP) antennapedia (Antp) recombinantly fused to a dominant-negative truncated mastermind-like protein (DN-MAML) called TR4. We have also developed a method of incorporating CPP to deliver intracellular antibody fragments against nuclear targets such as Notch and mastermind. Results: The Antp-DN-MAML protein (TR4) has similar potency to many of the gamma-secretase inhibitors being developed against this pathway but more specific for the NTC.TR4 can inhibit tumour cells in vitro and in vivo and provide some insight into the mechanism of action. Also such intracellular targets are accessible to antibodies, provided they are delivered using CPP such as antennapedia. Conclusions: .As a model system, scFvs have been raised to key epitopes in the NTC which can lead to complex inhibition and the same downstream effects as TR4.In addition, we further demonstrate that there is a more general approach to trascription factor inhibition using single-chain Fvs fused to Antp

Cutaneous β-human papillomavirus E6 proteins bind Mastermind-like coactivators and repress Notch signaling

The Notch signaling pathway is a key determinant in keratinocyte differentiation and growth cycle arrest, and has been reported to have a tumor suppressor function in skin. The papillomavirus life cycle is intricately linked to the differentiation status of keratinocytes. Papillomaviruses are associated with benign proliferative epithelial lesions in their respective hosts. Although human papillomaviruses (HPVs) associated with genital tract lesions have been extensively studied, studies of the cutaneous HPVs are more limited. In particular, it is well established that the E6 proteins of high-risk HPVs of the α-genus such as HPV16 and HPV18 mediate the degradation of p53 by its association with the ubiquitin ligase E6AP. In contrast, less is known about the cellular activities of the cutaneous HPVs of the β-genus. By using an unbiased proteomic approach, we identify MAML1 and other members of the Notch transcription complex as high-confidence cellular interacting proteins of E6 proteins of the β-genus HPVs and of the bovine papillomavirus type 1 associated with cutaneous fibropapillomas. We show that bovine papillomavirus type 1 and β-HPV E6 repress Notch transcriptional activation, and that this repression is dependent on an interaction with MAML1. Finally, we show that the expression levels of endogenous Notch target genes are repressed by β-HPV E6 proteins. These findings elucidate a mechanism of viral antagonism of Notch signaling, and suggest that Notch signaling is an important epithelial cell pathway target for the β-HPVs.

Conformational Locking upon Cooperative Assembly of Notch Transcription Complexes

The Notch intracellular domain (NICD) forms a transcriptional activation complex with the DNA-binding factor CSL and a transcriptional co-activator of the Mastermind family (MAML). The "RAM" region of NICD recruits Notch to CSL, facilitating the binding of MAML at the interface between the ankyrin (ANK) repeat domain of NICD and CSL. Here, we report the X-ray structure of a human MAML1/RAM/ANK/CSL/DNA complex, and probe changes in component dynamics upon stepwise assembly ofa MAML1/NICD/CSL complex using HX-MS. Association of CSL with NICD exerts remarkably little effect on the exchange kinetics of the ANK domain, whereas MAML1 binding greatly retards the exchange kinetics of ANK repeats 2-3. These exchange patterns identify critical features contributing to the cooperative assembly of Notch transcription complexes (NTCs), highlight the importance of MAML recruitment in rigidifying the ANK domain and stabilizing its interface with CSL, and rationalize the requirement for MAML1 in driving cooperative dimerization of NTCs on paired-site DNA.

In Notch, One ANK Repeat Is Not Like the Other

In this issue of Structure, Choi etal. use hydrogen/deuterium exchange mass spectrometry to characterize a Notch transcription complex (NTC). When interpreted in the context of NTC X-ray structures, their findings reveal important molecular insights into the dynamics that underlie complex assembly.

Ubiquitination of Notch1 is regulated by MAML1-mediated p300 acetylation of Notch1

Earlier studies demonstrated the involvement of the p300 histone acetyltransferase in Notch signaling but the precise mechanisms by which p300 might modulate Notch function remains to be investigated. In this study, we show that p300 acetylates Notch1 ICD in cell culture assay and in vitro, and conserved lysines located within the Notch C-terminal nuclear localization signal are essential for Notch acetylation. MAML1 and CSL, which are components of the Notch transcription complex, enhance Notch acetylation and we suggest that MAML1 increases Notch acetylation by potentiating p300 autoacetylation. Furthermore, MAML1-dependent acetylation of Notch1 ICD by p300 decreases the ubiquitination of Notch1 ICD in cellular assays. CDK8 has been shown to target Notch1 for ubiquitination and proteosomal degradation. We show that CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300. Therefore, we speculate that acetylation of Notch1 might be a mechanism to regulate Notch activity by interfering with ubiquitin-dependent pathways.

Probing the druggability of protein–protein interactions: targeting the Notch1 receptor ankyrin domain using a fragment-based approach

In order to achieve greater selectivity in drug discovery, researchers in both academia and industry are targeting cell regulatory systems. This often involves targeting the protein-protein interactions of regulatory multiprotein assemblies. Protein-protein interfaces are widely recognized to be challenging targets as they tend to be large and relatively flat, and therefore usually do not have the concave binding sites that characterize the so-called 'druggable genome'. One such prototypic multiprotein target is the Notch transcription complex, where an extensive network of protein-protein interactions stabilize the ternary complex comprising the ankyrin domain, CSL (CBF1/suppressor of Hairless/Lag-1) and MAML (Mastermind-like). Enhanced Notch activity is implicated in the development of T-ALL (T-cell acute lymphoblastic leukaemia) and selective inhibitors of Notch would be useful cancer medicines. In the present paper, we describe a fragment-based approach to explore the druggability of the ankyrin domain. Using biophysical methods and X-ray crystal structure analyses, we demonstrate that molecules can bind to the surface of the ankyrin domain at the interface region with CSL and MAML. We show that they probably represent starting points for designing larger compounds that can inhibit important protein-protein interactions that stabilize the Notch complex. Given the relatively featureless topography of the ankyrin domain, this unexpected development should encourage others to explore the druggability of such challenging multiprotein systems using fragment-based approaches.

MAML1 regulates cell viability via the NF-κB pathway in cervical cancer cell lines

The Notch signaling pathway plays important roles in tumorigenesis in a context-dependent manner. In human cervical cancer, alterations in Notch signaling have been reported, and both tumor-suppressing and tumor-promoting roles of Notch signaling have been proposed; however, the precise molecular mechanisms governing these roles in cervical cancer remain controversial. MAML is a transcriptional co-activator originally identified by its role in Notch signaling. Recent evidence suggests that it also plays a role in other signaling pathways, such as the p53 and β-catenin pathways. MAML is required for stable formation of Notch transcriptional complexes at the promoters of Notch target genes. Chromosomal translocations affecting MAML have been shown to promote tumorigenesis. In this study, we used a truncated dominant-negative MAML1 (DN-MAML) to investigate the role of MAML in HPV-positive cervical cancer cell lines. Three human cervical cancer cell lines (HeLa, SiHa and CaSki) expressed all Notch receptors and the Notch target genes Hes1 and MAML1. Among these 3 cell lines, constitutive appearance of cleaved Notch1 was found only in CaSki cells, which suggests that Notch1 is constitutively activated in this cell line. Gamma secretase inhibitor (GSI) treatment, which suppresses Notch receptor activation, completely abrogated this form of Notch1 but had no effect on cell viability. Overexpression of DN-MAML by retroviral transduction in CaSki cells resulted in significant decreases in the mRNA levels of Hes1 and Notch1 but had no effects on the levels of MAML1, p53 or HPV E6/E7. DN-MAML expression induced increased viability of CaSki cells without any effect on cell cycle progression or cell proliferation. In addition, clonogenic assay experiments revealed that overexpression of DN-MAML resulted in increased colony formation compared to the overexpression of the control vector. When the status of the NF-κB pathway was investigated, CaSki cells overexpressing DN-MAML exhibited loss of phospho-IκBα, decreased total IκBα and nuclear localization of NF-κB p65, which suggests that the NF-κB pathway is hyperactivated. Furthermore, increased level of cleaved Notch1 was detected when DN-MAML was expressed. When DN-MAML-overexpressing cells were treated with GSI, significantly decreased cell viability was observed, indicating that inhibition of Notch signaling using GSI treatment and DN-MAML expression negatively affects cell viability. Taken together, targeting Notch signaling using DN-MAML and GSI treatment may present a novel method to control cell viability in cervical cancer cells.

Assembly of a Notch Transcriptional Activation Complex Requires Multimerization

Notch transmembrane receptors direct essential cellular processes, such as proliferation and differentiation, through direct cell-to-cell interactions. Inappropriate release of the intracellular domain of Notch (N(ICD)) from the plasma membrane results in the accumulation of deregulated nuclear N(ICD) that has been linked to human cancers, notably T-cell acute lymphoblastic leukemia (T-ALL). Nuclear N(ICD) forms a transcriptional activation complex by interacting with the coactivator protein Mastermind-like 1 and the DNA binding protein CSL (for CBF-1/Suppressor of Hairless/Lag-1) to regulate target gene expression. Although it is well understood that N(ICD) forms a transcriptional activation complex, little is known about how the complex is assembled. In this study, we demonstrate that N(ICD) multimerizes and that these multimers function as precursors for the stepwise assembly of the Notch activation complex. Importantly, we demonstrate that the assembly is mediated by N(ICD) multimers interacting with Skip and Mastermind. These interactions form a preactivation complex that is then resolved by CSL to form the Notch transcriptional activation complex on DNA.