Constitutive Activation Of Notch Signaling Research Articles

Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma.

Human dedifferentiated liposarcoma (DDLPS) is a rare but lethal cancer with no driver mutations being identified, hampering the development of targeted therapies. We and others recently reported that constitutive activation of Notch signaling through overexpression of the Notch1 intracellular domain (NICDOE) in murine adipocytes leads to tumors resembling human DDLPS. However, the mechanisms underlying the oncogenic functions of Notch activation in DDLPS remains unclear. Here, we show that Notch signaling is activated in a subset of human DDLPS and correlates with poor prognosis and expression of MDM2, a defining marker of DDLPS. Metabolic analyses reveal that murine NICDOE DDLPS cells exhibit markedly reduced mitochondrial respiration and increased glycolysis, mimicking the Warburg effect. This metabolic switch is associated with diminished expression of peroxisome proliferator-activated receptor gamma coactivator 1α (Ppargc1a, encoding PGC-1α protein), a master regulator of mitochondrial biogenesis. Genetic ablation of the NICDOE cassette rescues the expression of PGC-1α and mitochondrial respiration. Similarly, overexpression of PGC-1α is sufficient to rescue mitochondria biogenesis, inhibit the growth and promote adipogenic differentiation of DDLPS cells. Together, these data demonstrate that Notch activation inhibits PGC-1α to suppress mitochondrial biogenesis and drive a metabolic switch in DDLPS.

Fibrous Caps in Atherosclerosis Form by Notch-Dependent Mechanisms Common to Arterial Media Development.

Atheromatous fibrous caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We tested whether the recruitment mechanisms are the same as in embryonic artery development, which relies prominently on Notch signaling to form the subendothelial medial SMC layers. Notch elements were expressed in regions of fibrous cap in human and mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice where the Notch pathway was inactivated in SMCs by conditional knockout of the essential effector transcription factor RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). The recruitment of cap SMCs was significantly reduced without major effects on plaque size. Lineage tracing revealed the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. Conversely, to analyze whether the loss of Notch signaling is required for SMC-derived cells to accumulate in atherogenesis, we studied atherosclerosis in mice with constitutive activation of Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development. Sequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the cap forms as a neo-media that restores the connection between endothelium and subendothelial SMCs, transiently disrupted in early atherogenesis.

Icariin promotes osteogenic differentiation by suppressing Notch signaling

Osteoporosis is a bone disease characterized by microarchitectural deterioration, low bone mass, and increased risk of fractures. Icariin (ICA), an active flavonoid glucoside isolated from Herba epimedii (HEF), is a potent stimulator of osteogenic differentiation and has potential applications for preventing bone loss in postmenopausal women. However, the molecular mechanism underlying the osteogenic effect of ICA has not yet been fully elucidated. In this study, we report that ICA treatment significantly elevated gene expression of osteogenic markers and increased alkaline phosphatase (ALP) activity in MC3T3-E1 and C3H10T1/2 cells. RNA sequencing revealed that the expression of several genes involved in the Notch pathway was decreased following ICA treatment. Real-time PCR further demonstrated that the mRNA levels of Notch ligands Jagged-1 (Jag1), lunatic fringe (Lfng), and Notch signaling downstream target gene Hey-1 were significantly decreased following ICA treatment. In addition, we found that constitutive activation of Notch signaling through overexpression of the intracellular domain of Notch (NICD) fully blocked ICA-induced osteoblast differentiation. Moreover, inhibiting Notch signaling with DAPT markedly enhanced osteogenic differentiation following ICA treatment. We found that the mRNA levels of Notch pathway molecules (Lfng, Notch1, Rbpjk and Nfatc1) were increased in ovariectomized (OVX) mice, and administration of ICA significantly decreased the expression of these genes. Our results suggest that ICA promotes osteogenic differentiation in vitro and alleviates osteoporosis in vivo through inhibition of the Notch signaling pathway.

Abstract 4636: Oncogenic Notch triggers neoplastic-tumorigenesis in a transition-zone like tissue microenvironment

Abstract The “seed and soil” hypothesis proposed by Stephen Paget (1889) highlights the importance of tissue microenvironment for secondary tumor formation. The microenvironment that promotes primary tumor origination, however, remains largely unclear. Transition zones, where two types of epithelial tissue meet, have been characterized as high-risk sites for tumorigenesis. Here we show that the Drosophila salivary gland (SG) imaginal ring (ImR) can be used as a model to study tumorigenesis in transition zones. We found that constitutive activation of Notch signaling during the third larval instar drives overproliferation and tumor formation in the SG ImR. Interestingly, tumorigenesis always occurs at the posterior end of the ImR, which is a transitional area between the polyploid giant cells and diploid ImR cells. These Notch-induced tumors display disrupted epithelial organization, grow continuously and can metastasize once transplanted into the abdomen of adult flies, suggesting that they have the characteristics of malignant neoplasms. Further analyses revealed that the SG ImR transition zone possesses endogenously high levels of Janus kinase/signal transducers and activators of transcription (JAK-STAT) and c-Jun amino-terminal kinases (JNK), both of which are necessary for tumor growth. In this region, JNK signaling induces the expression of matrix metalloproteinase 1 (MMP1), which is also required for tumor formation in this model system. Furthermore, we found that ectopic MMP1 expression can transform the anterior end of the SG ImR, which is normally refractory to oncogenic Notch-induced tumorigenesis, into a tumor hotspot. Together, these studies reveal that local endogenous activation of JNK and JAK-STAT signaling creates a niche-like tissue microenvironment that is susceptible to oncogene-induced neoplastic tumor formation. Citation Format: Wu-Min Deng, Sheng-An Yang. Oncogenic Notch triggers neoplastic-tumorigenesis in a transition-zone like tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4636.

Notch signaling regulates cell density-dependent apoptosis of NIH 3T3 through an IL-6/STAT3 dependent mechanism

Apoptosis is a physiological process that plays a critical maintenance role in cellular homeostasis. Previous reports have demonstrated that cells undergo apoptosis in a cell density-dependent manner, which is regulated, in part, by signal transducers and activators of transcription (STAT) 3. The molecular mechanisms regulating cell density-dependent apoptosis, however, has not been thoroughly investigated to date. Since Notch signaling is activated via direct cell-to-cell contact and plays a pivotal role in cell fate decisions, we examined the role of Notch signaling in cell density-dependent apoptosis of mouse embryonic fibroblasts NIH 3T3 cells. With the increase in cell density, IL-6 expression was induced, which was necessary for STAT3 activation as well as apoptosis regulation. Notch signaling was also activated in a cell-density dependent manner. Blocking Notch signaling either through siRNA-mediated targeting of Jagged1 expression or γ-secretase inhibitor treatment demonstrated that Notch signaling activation was necessary for IL-6 induction. Constitutive activation of Notch signaling via the overexpression of Notch1 intracellular domain was sufficient for the induction of IL-6, which was mediated via direct transcriptional activation. Taken together, our study indicates that Notch signaling regulates cell density-dependent apoptosis through IL-6/STAT3-dependent mechanism. Consequently, Notch signaling might represent a novel therapeutic target in diseases characterized by dysregulated apoptosis.

Inhibition of Delta-induced Notch signaling using fucose analogs.

Notch is a cell-surface receptor that controls cell fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools for inhibiting Notch activity are being developed as cancer therapeutics. Towards this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.

Oleanolic Acid Enhances Mesenchymal Stromal Cell Osteogenic Potential by Inhibition of Notch Signaling

Oleanolic acid (OA), a pentacyclic triterpenoid, has been shown to modulate multiple signaling pathways in a variety of cell linages. But the mechanisms underlying OA-mediated mesenchymal stromal cell (MSC) osteogenic differentiation are not known. In this study, we examined effects of OA on cell viability, osteogenic differentiation in MSCs, and the involvement of Notch and BMP signaling. OA induced bone marrow derived MSC differentiation towards osteoprogenitor cells and inhibited Notch signaling in a dose dependent manner. Constitutive activation of Notch signaling fully blocked OA induced MSC osteogenic differentiation. The expression level of early osteogenic marker genes, ALP, Runx2, and type I collagen, which play a critical role in MSC to osteoblast transition and servers as a downstream target of BMP signaling, was significantly induced by OA. Furthermore, BMP2 mediated MSC osteogenic differentiation was significantly enhance by OA treatment, indicating a synergistic effect between BMP2 and OA. Our results suggest that OA is a promising bioactive agent for bone tissue regeneration, and inhibition of Notch signaling is required for its osteogenic effects on MSCs.

Abstract 4623: Reevaluation of the role of Notch signaling in melanoma tumor development, melanoma cell survival and drug resistance

Abstract Notch is one of the simplest signaling pathways that is required for almost every cell type in the body. It plays several roles such as cell fate decision, differentiation and stem cell maintenance. Notch signaling plays a role in the maintenance of melanoblasts and melanocyte stem cells of the epidermis by preventing their apoptosis. Notch signaling has also been implicated in different aspects of melanoma biology including tumor initiation, progression and metastasis. Accordingly, Notch has been targeted for melanoma therapy both in preclinical xenograft models as well as clinical trials. Notch inhibitors, mainly γ-secretase inhibitors, have been widely used for this purpose. However, clinical trials with these agents have not been proven to be highly effective for melanoma treatment. In previously published studies we showed that Notch signaling is involved in regulation of melanoma trans-differentiation, a prognostic feature in primary melanoma. In this study, we reevaluated the role of Notch in melanoma development, cell survival and sensitivity to BRAF and MEK inhibitors. First, our preliminary data using the BRAFV600E/Pten mouse model showed that topical application of γ-secretase inhibitor DAPT delays tumor formation and growth. In several early passage metastatic human melanoma cell lines, we found that Notch receptors (Notch 1-3) are highly upregulated compared to normal epidermal melanocytes. Interestingly, mRNA levels for Notch1-3 receptors in normal melanocytes were either higher or comparable to their levels in the cell lines. This suggested that Notch receptor levels are tightly regulated in normal melanocytes by post-transcriptional mechanisms. Presence of cleaved Notch intracellular domain (NICD), both cytoplasmic and nuclear, in melanoma cells, but not in melanocytes, indicated constitutive activation of Notch signaling in melanoma cells. However, inhibition of Notch activation by treatment with γ-secretase inhibitor had no significant effect on survival of melanoma cells in vitro. Thus, Notch signaling appears to be dispensable for melanoma cell survival. Additionally, inhibition of Notch activation did not alter the sensitivity of melanoma cells to BRAF(V600E) and MEK inhibitors. Our results highlight the need for re-examination of Notch-regulated melanoma trans-differentiation as a therapeutic outcome. Citation Format: Dareen Mikheil, Carlos Rodriguez, Ashika Jayanthi, Prithvi Yarlagadda, Vijaysaradhi Setaluri. Reevaluation of the role of Notch signaling in melanoma tumor development, melanoma cell survival and drug resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4623.

Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling.

T and innate lymphoid cells (ILCs) share some aspects of their developmental programs. However, although Notch signaling is strictly required for T cell development, it is dispensable for fetal ILC development. Constitutive activation of Notch signaling, at the common lymphoid progenitor stage, drives T cell development and abrogates ILC development by preventing Id2 expression. By combining single-cell transcriptomics and clonal culture strategies, we characterize two heterogeneous α4β7-expressing lymphoid progenitor compartments. αLP1 (Flt3(+)) still retains T cell potential and comprises the global ILC progenitor, while αLP2 (Flt3(-)) consists of ILC precursors that are primed toward the different ILC lineages. Only a subset of αLP2 precursors is sensitive to Notch signaling required for their proliferation. Our study identifies, in a refined manner, the diversity of transitional stages of ILC development, their transcriptional signatures, and their differential dependence on Notch signaling.

An oncogenic enhancer enemy (N-Me) in T-ALL

Constitutive activation of NOTCH signaling plays a central role in the pathogenesis of over 60% of T-cell acute lymphoblastic leukemias (T-ALL) harboring activating mutations in NOTCH1 or disruption of FBXW7, a critical negative regulator of NOTCH signaling.1 Expression of oncogenic NOTCH1 drives T-cell transformation activating a transcriptional program that includes multiple genes promoting leukemia cell growth and proliferation. Among these, the MYC oncogene has been highlighted as an important mediator of NOTCH1 induced transformation as NOTCH1 signaling induces transcriptional upregulation of MYC expression and NOTCH1 and MYC share multiple common target genes involved in leukemia cell growth.2 Still, demonstrating direct regulation of MYC by NOTCH1 has remained elusive as NOTCH1 fails to activate MYC promoter activity in reporter assays. This enigmatic link between NOTCH1 and MYC in T-ALL has been now clarified with the identification of N-Me (for NOTCH MYC enhancer), a long-range distal enhancer controlling MYC expression and T-cell transformation downstream of NOTCH1 (Fig. 1).3 The N-Me enhancer is located +1,427 kb from MYC in a large gene desert region extending telomeric from the MYC locus and represents the strongest peak of NOTCH1 binding in ChIP-seq analysis of T-ALL cells. Notably, and despite its distal genomic location, this regulatory element directly interacts with and controls MYC promoter activity as demonstrated by chromatin configuration 3C analysis and reporter assays. The functional importance of N-Me in the control of MYC expression is further supported by multispecies DNA sequence homology analyses which revealed remarkable conservation of this region in mammals, birds and reptiles. In addition, analysis of functional chromatin marks showed bona fide active enhancer features including P300 occupancy, high levels of H3K4me1 and low levels of H3K4me3 associated with N-Me. Moreover, detailed epigenetic profiling across 64 haematopoietic and non-haematopoietic cell lines and tissues revealed that this regulatory element is located within a major super-enhancer specifically active in T-cells. Consistently with these results, analysis of N-Me knockout and conditional knockout mice demonstrated a critical role for this long-range enhancer in T-cell development and NOTCH1-induced T-ALL.3 Thus, N-Me deficient mice showed marked thymic atrophy with depletion of CD4 CD8 double positive thymocytes. This phenotype was cell autonomous and was associated with decreased Myc expression in DN3, DN4 and ISP T-cell progenitors. Importantly, these T-cell developmental defects recapitulate those observed in T-cell specific Myc knockout mice,4 and were rescued by retroviral expression of MYC, formally demonstrating a strict requirement for N-Me mediated Myc expression in T-cell development. In addition, the NOTCH-N-Me-Myc regulatory axis showed to be essential for NOTCH1 induced T-ALL. Thus, N-Me knockout bone marrow progenitor cells failed to generate leukemia upon retroviral expression of oncogenic NOTCH1, and secondary deletion of N-Me in fully established NOTCH1-induced T-ALL resulted in profound antileukemic effects and almost complete suppression of leukemia initiating cell activity. Strikingly, loss of one copy of N-Me showed intermediate phenotypes including partial reduction of thymocyte numbers; reduced penetrance of T-ALL upon oncogenic NOTCH1 activation; and delayed tumor progression of fully established NOTCH1-driven tumors. These results highlight the exact dose-dependent function of the NOTCH1-NMe-Myc regulatory circuitry. Finally, genomic profiling of human leukemias identified recurrent focal duplications encompassing the N-Me enhancer in T-ALL supporting that increased N-Me dosage may contribute to T-cell transformation by strengthening NOTCH1 driven MYC expression. Figure 1. Schematic representation of N-Me activity during thymocyte development and in NOTCH1-induced T-ALL. Overall our results highlight the importance of long-range enhancers involved in the control of MYC expression in the pathogenesis of human cancer and are in line with previous results in myeloid leukemia and colon cancer.5,6 Still, the detailed characterization of the N-Me enhancer opens new outstanding questions that remain to be addressed. Most intriguingly, NOTCH1 and MYC play important developmental roles in multiple tissues other than T-cells and NOTCH signaling regulation of MYC expression has been implicated in the pathogenesis of breast cancer and other solid tumors;7,8 however N-Me seems to be strictly T-cell specific, suggesting that other as yet unidentified NOTCH-controlled MYC enhancers may be responsible for MYC expression outside T-cells. In addition, it is unclear what factors drive the T-cell specific activity of the N-Me enhancer. Finally, it has not escaped our attention that our identification of N-Me duplications in T-ALL support a potentially important and broader role for chromosomal copy number alterations and mutations occurring in intergenic regulatory elements in the pathogenesis of human cancer.

SKP2 Is Dispensable for Normal T-Cell Development but Required for T-Cell Leukemogenesis

Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer. Despite the significant clinical successes in the treatment of pediatric T-ALL, leukemia relapse, refractory disease and induction failure (around 30% of patients) remain significant clinical problems, which are often life-threatening. ALL remains the second leading cause of childhood death. Thus, more effective, curative therapeutic strategies are much needed, particularly for refractory and relapse T- ALL. Recent advances in understanding the biology and the molecular alterations of acute lymphoblastic leukemias have led to identification of new molecular targets, such as the Notch signaling pathway. Constitutive activation of Notch signaling is involved in more than 50% of human T-ALL, and overexpression of activated Notch induces T-cell leukemia and lymphoma in murine tumor models. However, disruption of Notch signaling by gamma-secretase inhibitors (GSI) failed to fulfill its clinical promise and, overall, the significant advances attained in dissecting the molecular effectors in T-ALL has yet to translate into effective, curative molecular therapies for relapse patients. Furthermore, despite multiple studies on Notch signaling, little is known on the role of its downstream mediators in T-cell ALL.Previous studies in our laboratory demonstrated that Notch1 activation induces transcriptional activation of SKP2, the F-box protein of the SCF E3-ubiquitin ligase complex. SKP2 is the main F-box protein regulating cell cycle, promoting downregulation of the CKIs (p21Cip1, p27Kip1, p57Kip2 and p130) and its overexpression accelerate cell cycle progression in hematopoietic cells. SKP2 overexpression is frequently associated with cancers, in particular lymphomas and leukemias, and correlates with poor prognosis.We found that Skp2 expression is dynamically regulated during T-cell development coinciding with the Notch expression pattern. Moreover, primary thymocytes cultured in vitro, responded to Notch stimulation by the Delta1 ligand increasing their Skp2 expression and their cell cycle status, whereas the loss of SKP2 impaired their ability to mount a proliferative response to IL-7 stimulation. Importantly, we observed that SKP2 expression is increased in T-ALL patient samples and that mice with Notch-induced T-cell leukemia showed 5 fold upregulation of Skp2 expression. Our hypotheses are that Notch activation promotes T-cell leukemogenesis by altering the cell cycle control through upregulation of Skp2, and that selective targeting of SKP2 is a novel, effective therapeutic strategy for childhood T-ALL. To test whether SKP2 is a key downstream mediator of Notch in T-ALL, we transduced oncogenic Notch (ICN; the constitutive intracellular form) in hematopoietic cells lacking SKP2 from Skp2-/- null mice, and in controls and we determine their ability to induce leukemia in irradiated recipients. Loss of SKP2 significantly delayed the development of T-cell leukemia and increased animal survival by 40%. Taken together, these results demonstrate a previously unrecognized role for SKP2 in the initiation and progression of T-ALL and its potential role as a therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity.

Beige adipocytes in white adipose tissue (WAT) are similar to classical brown adipocytes in that they can burn lipids to produce heat. Thus, an increase in beige adipocyte content in WAT browning would raise energy expenditure and reduce adiposity. Here we report that adipose-specific inactivation of Notch1 or its signaling mediator Rbpj in mice results in browning of WAT and elevated expression of uncoupling protein 1 (Ucp1), a key regulator of thermogenesis. Consequently, as compared to wild-type mice, Notch mutants exhibit elevated energy expenditure, better glucose tolerance and improved insulin sensitivity and are more resistant to high fat diet-induced obesity. By contrast, adipose-specific activation of Notch1 leads to the opposite phenotypes. At the molecular level, constitutive activation of Notch signaling inhibits, whereas Notch inhibition induces, Ppargc1a and Prdm16 transcription in white adipocytes. Notably, pharmacological inhibition of Notch signaling in obese mice ameliorates obesity, reduces blood glucose and increases Ucp1 expression in white fat. Therefore, Notch signaling may be therapeutically targeted to treat obesity and type 2 diabetes.

Abstract A226: Identification of new synergistic compounds for the treatment of NOTCH-driven T-ALL.

Abstract T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 15% of childhood and 25% of adult ALLs. Constitutive activation of NOTCH signaling as result of activating mutations in the NOTCH1 gene is a hallmark of this disease and small molecule gamma-secretase inhibitors, which effectively block activation of the NOTCH1 receptor, have been proposed as targeted therapy. However, the progress of NOTCH1-blocking agents such as in the clinic has been limited. Here we hypothesized that detailed transcriptional profiling of T-ALL lymphoblasts upon inhibition of NOTCH1 signaling would uncover major effector pathways downstream of NOTCH1. Moreover, we proposed that these signatures would facilitate the identification of new drugs and drug combinations for the treatment of T-ALL. Gene expression profiling of NOTCH1 induced leukemias treated with DBZ, a highly active gamma-secretase inhibitor, identified numerous genes regulated by NOTCH1 in human leukemia. Notably these signatures were enriched in NOTCH1 direct target genes such as Hes1 and revealed a prominent role of NOTCH signaling in the control of cellular metabolic pathways. Following on these results we performed Connectiviy map analysis to identify drugs whose transcriptional effects could antagonize those of oncogenic NOTCH1 in human leukemia. This analysis identified HDAC inhibitors, anti-psychotic agents, anti-malarials and compounds targeting the PI3K and NFKB signaling pathways. Detailed analysis of the effects of these drugs alone and in combination with DBZ demonstrated strong antileukemic activity and strong synergism when combined with inhibition of NOTCH signaling with a gamma-secretase inhibitor. Moreover, biochemical and transcriptional profiling of these drugs identifies agents with unique and related mechanisms of action. Overall these results identify new antileukemic drugs with strongly synergistic activity in combination with NOTCH1 inhibition for the treatment of T-ALL Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A226. Citation Format: Marta Sanchez Martin, Alberto Ambesi, Daniel Herranz, Adolfo Ferrando. Identification of new synergistic compounds for the treatment of NOTCH-driven T-ALL. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A226.

Akt1 Mediates Neuronal Differentiation in Zebrafish via a Reciprocal Interaction with Notch Signaling

Akt1 is well known for its role in regulating cell proliferation, differentiation, and apoptosis and is implicated in tumors and several neurological disorders. However, the role of Akt1 in neural development has not been well defined. We have isolated zebrafish akt1 and shown that this gene is primarily transcribed in the developing nervous system, and its spatiotemporal expression pattern suggests a role in neural differentiation. Injection of akt1 morpholinos resulted in loss of neuronal precursors with a concomitant increase in post-mitotic neurons, indicating that knockdown of Akt1 is sufficient to cause premature differentiation of neurons. A similar phenotype was observed in embryos deficient for Notch signaling. Both the ligand (deltaA) and the downstream target of Notch (her8a) were downregulated in akt1 morphants, indicating that Akt1 is required for Delta-Notch signaling. Furthermore, akt1 expression was downregulated in Delta-Notch signaling-deficient embryos and could be induced by constitutive activation of Notch signaling. In addition, knockdown of Akt1 was able to nullify the inhibition of neuronal differentiation caused by constitutive activation of Notch signaling. Taken together, these results provide in vivo evidence that Akt1 interacts with Notch signaling reciprocally and provide an explanation of why Akt1 is essential for the inhibition of neuronal differentiation.

DDX5 is a positive regulator of oncogenic NOTCH1 signaling in T cell acute lymphoblastic leukemia

Notch signaling is a highly conserved cell-cell communication pathway regulating normal development and tissue homeostasis. Aberrant Notch signaling represents an important oncogenic mechanism for T cell acute lymphoblastic leukemia (T-ALL), an aggressive subset of the most common malignant childhood cancer ALL. Therefore, understanding the molecular regulation of Notch signaling is critical to identify new approaches to block aberrant Notch oncogenic activity. The family of three MAML transcriptional coactivators is crucial for Notch signaling activation. The prototypic member MAML1 is the major coactivator that regulates Notch oncogenic activities in leukemic cells. However, the molecular basis underlying MAML1 coactivator function that contributes to Notch signaling remains unclear. In this study, we performed proteomic studies and identified DDX5, an ATP-dependent DEAD-box RNA helicase, as a component of the MAML1 protein complex. DDX5 interacts with MAML1 in vitro and in vivo, and is associated with the endogenous NOTCH1 transcription activation complex in human T-ALL leukemic cells. Lentivirus-mediated short-hairpin RNA knock-down of DDX5 resulted in decreased expression of Notch target genes, reduced cell proliferation and increased apoptosis in cultured human leukemic cells with constitutive activation of Notch signaling. Also, DDX5 depletion inhibited the growth of human leukemia xenograft in nude mice. Moreover, DDX5 is highly expressed in primary human T-ALL leukemic cells based on the analyses of Oncomine and GEO databases, and Immunohistochemical staining. Our overall findings revealed a critical role of DDX5 in promoting efficient Notch-mediated transcription in leukemic cells, suggesting that DDX5 might be critical for NOTCH1-mediated T-ALL pathogenesis and thus is a potential new target for modulating the Notch signaling in leukemia.

The molecular basis of T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemias (T-ALLs) arise from the malignant transformation of hematopoietic progenitors primed toward T cell development, as result of a multistep oncogenic process involving constitutive activation of NOTCH signaling and genetic alterations in transcription factors, signaling oncogenes, and tumor suppressors. Notably, these genetic alterations define distinct molecular groups of T-ALL with specific gene expression signatures and clinicobiological features. This review summarizes recent advances in our understanding of the molecular genetics of T-ALL.

Notch signaling mediates TGF-β1-induced epithelial–mesenchymal transition through the induction of Snai1

Epithelial–mesenchymal transition (EMT) is a process by which epithelial cells undergo phenotypic transition to mesenchymal cells and thus is involved in the pathogenesis of tumor metastasis and organ fibrosis. Notch signaling is a highly conserved pathway that regulates intercellular communication and directs cell fate decisions. Here, we show the critical role of Notch signaling in TGF-β1-induced EMT. Inhibition of Notch signaling either by γ-secretase inhibitor or by knocking down of Notch signaling molecules by small interfering RNA abrogated EMT in association with decreased expression of Snai1. Constitutive activation of Notch signaling was sufficient for the induction of Snai1 as well as Notch ligand Jagged1. Notch signaling induced Snai1 expression via direct transcriptional activation. Collectively, these data show that Notch signaling activation promote TGF-β1-induced EMT through the induction of Snai1. Further studies on Notch signaling may provide diagnostic and therapeutic targets for cancer and fibrotic disease.

Current views on the role of Notch signaling and the pathogenesis of human leukemia

The Notch signaling pathway is highly conserved from Drosophila to humans and plays an important role in the regulation of cellular proliferation, differentiation and apoptosis.Constitutive activation of Notch signaling has been shown to result in excessive cellular proliferation and a wide range of malignancies, including leukemia, glioblastoma and lung and breast cancers. Notch can also act as a tumor suppressor, and its inactivation has been associated with an increased risk of spontaneous squamous cell carcinoma. This minireview focuses on recent advances related to the mechanisms and roles of activated Notch1, Notch2, Notch3 and Notch4 signaling in human lymphocytic leukemia, myeloid leukemia and B cell lymphoma, as well as their significance, and recent advances in Notch-targeted therapies.

Abstract 3388: DDX5 modulates Notch signaling via interaction with the MAML1 co-activator and is required to optimal Notch signaling in leukemic cells

Abstract Notch receptor-mediated signaling is essential for embryonic development and adult tissue homeostasis by regulating cell proliferation, differentiation and survival. Aberrant Notch signaling is linked to many epithelial cancers and hematological malignancies. For example, activated NOTCH1 mutations are detected in more than 50% of T cell acute lymphoblastic leukemia (T-ALL) patients. Since the constitutive activation of Notch signaling results in leukemic cell transformation and is required for the maintenance of the leukemic phenotype, the Notch pathway is a promising and highly specific molecular therapeutic target for T-ALL. We have previously identified the family of the MAML transcriptional co-activators as essential components of the core Notch transcription activation complex. The prototypic member MAML1 was found to be the major co-activator that regulates NOTCH1 oncogenic activities in leukemic cells. Hence, the modulation of MAML1 expression levels or functional activities is expected to affect Notch-induced leukemic initiation and progression. However, it remains unclear how MAML1 function is regulated. To gain insights into the molecular basis underlying MAML1 function, we undertook the task of identifying cellular factors that interact with MAML1 and characterizing their roles in regulating Notch signaling and Notch-mediated T cell leukemia. Through proteomic studies, we identified DDX5, an ATP-dependent DEAD-box RNA helicase, as a component in the MAML1 protein complex. Given the role of DDX5 as a transcriptional modulator, we reasoned that DDX5 might be a critical regulator of MAML1-mediated Notch target genes transcription. We showed that DDX5 interacts with MAML1 in vitro and in vivo, and is associated with the endogenous Notch1 transcription activation complex through interaction with MAML1. Lentiviral-mediated shRNA knockdown of DDX5 resulted in decreased expression of Notch target signature, along with reduced cell proliferation and survival in leukemic cells that carry activated NOTCH1 mutations. Our findings thus revealed an essential role of DDX5 in promoting efficient Notch-mediated transcription in leukemic cells where the Notch pathway is constitutively activated, suggesting that DDX5 might be critical for Notch-mediated T-ALL pathogenesis and could be a potential new target for modulating the Notch signaling in leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3388. doi:10.1158/1538-7445.AM2011-3388

Regulation of glioblastoma stem cells by retinoic acid: role for Notch pathway inhibition

It is necessary to understand mechanisms by which differentiating agents influence tumor-initiating cancer stem cells. Toward this end, we investigated the cellular and molecular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA). GBM-SCs were grown as non-adherent neurospheres in growth factor supplemented serum-free medium. RA treatment rapidly induced morphology changes, induced growth arrest at G1/G0 to S transition, decreased cyclin D1 expression and increased p27 expression. Immunofluorescence and western blot analysis indicated that RA induced the expression of lineage-specific differentiation markers Tuj1 and GFAP and reduced the expression of neural stem cell markers such as CD133, Msi-1, nestin and Sox-2. RA treatment dramatically decreased neurosphere-forming capacity, inhibited the ability of neurospheres to form colonies in soft agar and inhibited their capacity to propagate subcutaneous and intracranial xenografts. Expression microarray analysis identified ∼350 genes that were altered within 48 h of RA treatment. Affected pathways included retinoid signaling and metabolism, cell-cycle regulation, lineage determination, cell adhesion, cell-matrix interaction and cytoskeleton remodeling. Notch signaling was the most prominent of these RA-responsive pathways. Notch pathway downregulation was confirmed based on the downregulation of HES and HEY family members. Constitutive activation of Notch signaling with the Notch intracellular domain rescued GBM neurospheres from the RA-induced differentiation and stem cell depletion. Our findings identify mechanisms by which RA targets GBM-derived stem-like tumor-initiating cells and novel targets applicable to differentiation therapies for glioblastoma.