Notch Target Hes1 Research Articles

Glycogen Synthase Kinase-3 Inhibition Promotes Proliferation and Neuronal Differentiation of Human-Induced Pluripotent Stem Cell-Derived Neural Progenitors

Human-induced pluripotent stem cell-derived neural progenitors (hiPSC-NPs) have the ability to self-renew and differentiate into glial and neuronal lineages, which makes them an invaluable source in cell replacement therapy for neurological diseases. Therefore, their enhanced proliferation and neuronal differentiation are pivotal features that can be used in repairing neurological injuries. One of the main regulators of neural development is Wnt signaling, which results in the inhibition of glycogen synthase kinase 3 (GSK-3). Here, we assess the impact of GSK-3 inhibition by the small molecule CHIR99021 on the expansion and differentiation of hiPSC-NPs in an adherent condition and a defined medium. Cell proliferation analyses have revealed that inhibition of GSK-3 in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) increased the proliferation of hiPSC-NPs across 10 passages. The inhibition of β-catenin signaling by XAV and NOTCH signaling by DAPT reversed CHIR impact on hiPSC-NPs proliferation. The target genes of β-catenin, C-MYC and CYCLIN D1 as well as NOTCH target genes, HES1 and HES5 were upregulated. The treatment of NPs by CHIR in the absence of bFGF and EGF resulted in an increase of neuronal differentiation rather than proliferation by stabilization of β-catenin regardless of the NOTCH pathway. Thus, GSK-3 inhibition has been shown to promote proliferation of the NPs by activating β-catenin and NOTCH-related cell cycle genes in the presence of bFGF and EGF. Additionally, during GSK-3 inhibition, an absence of these growth factors allows for the switch to neuronal differentiation with a bias toward a dopaminergic fate. This may provide desired cells that can be used in therapeutic applications and offer insights into the etiology of some neurological disorders.

Hes1-mediated constraint of C/EBPα is essential for T-cell development (111.22)

Abstract Notch signaling in the thymus induces expression of T-cell genes and discourages progenitors from adopting alternative fates. Recent work has suggested that T-cell progenitors arrive at the thymus with T and non-T (B, NK) lymphoid potential as well as dendritic cell potential and a degree of myeloid potential; however, the importance of actively repressing alternative gene expression programs in T-cell development has not been adequately addressed. To examine the role of Notch-mediated gene repression in T-cell development, we used mice deficient for the Notch target and transcriptional repressor Hes1. We found that Hes1-deficient progenitors failed to make normal numbers of T-cells upon culture with Notch ligands, but instead failed to downregulate the myeloid transcription factor C/EBPα and gave rise to increased numbers of Mac1+ myeloid cells. To test whether the primary role of Hes1 in T-cell development is to repress expression of the myeloid transcription factor C/EBPα, we deleted floxed alleles of C/EBPα in Hes1-deficient progenitors using a retroviral Cre recombinase and co-cultured transduced progenitors with Notch-ligand expressing stromal cells. We found that deletion of C/EBPα restored T-cell development in Hes1-deficient progenitors. These results establish that an essential function of Hes1 is to constrain progenitors from activating myeloid lineage programs early in T-cell development.

251 A Functional Interplay Between TGF- and Notch Signaling Determines Esophageal Cancer Cell Fates

Background: Esophageal adenocarcinoma (EA) is often considered to arise from a clonal stem like population of cells, potentially responsible for its poor prognosis. TGF-β and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. We have demonstrated loss of TGF-β signaling components and activation of Notch signaling in Barrett's esophageal adenocarcinoma; however, their contributions to EA and the mechanisms of their action remain unclear. Methods: Immunoblotting and immunofluorescence were used to evaluate protein expression and localization. Notch targets Hes-1, SOX9 and C-MYC transcription were assayed using their luciferase reporters and Q-PCR. Functional studies were performed in β2SP wild type and mutant MEFs cells and in EA cell lines genetically engineered to express high or low levels of β2SP. Results: Increased levels of Notch signaling Hes1 and Jagged1 occurred in EA tissues and cell lines, compared to normal tissues. Loss of β2SP in MEFs cells increased Hes1 expression by 40 fold. In addition, SOX9, a target gene of Notch signaling and a documented stem cell marker was highly up-regulated in EA tumor cells and tissues. Expression of SOX9 was increased in the absence of β2SP in MEFs cells by 9 fold. Down-regulation of β2SP in SKTG-4, FLO-1 and BE3 EA cells by lentivirus shRNA led to increased SOX9 expression and enhanced nuclear localization of both active intracellular Notch1 domain (ICN1) and SOX9. Reintroducing β2SP into EA cells with knock down β2SP decreased SOX9 promoter activity by 10 fold. Concomitantly, an increased proportion of stem cells in β2SP knock down cells were identified using stem cell marker OCT3/4, indicating expansion of putative cancer stem cells in the absence of β2SP. Most interestingly, we observed a direct interaction between Smad3 and ICN1 via Smad3 MH1 domain by GST-pull down assays and that loss of β2SP increases the binding of Smad3 with ICN1 and induces Notch targets SOX9 and C-MYC transcription and decreased expression of its own targets P21, P27 and E-Cadherin. Conclusions: Our findings suggests that loss of TGF-β signaling adaptor β2SP may switch Smad3 function from tumor suppression to tumor promotion by binding intracellular Notch1 domain (ICN1) and activating Notch signaling. Thereby, a potential therapeutic value for targeted therapy in EA is in the setting of loss of β2SP/TGF-β with active Notch signaling.

252 CD44 Defines Highly Plastic Esophageal Squamous Cancer Cells With Tumor Initiating Capabilities

Background: Esophageal adenocarcinoma (EA) is often considered to arise from a clonal stem like population of cells, potentially responsible for its poor prognosis. TGF-β and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. We have demonstrated loss of TGF-β signaling components and activation of Notch signaling in Barrett's esophageal adenocarcinoma; however, their contributions to EA and the mechanisms of their action remain unclear. Methods: Immunoblotting and immunofluorescence were used to evaluate protein expression and localization. Notch targets Hes-1, SOX9 and C-MYC transcription were assayed using their luciferase reporters and Q-PCR. Functional studies were performed in β2SP wild type and mutant MEFs cells and in EA cell lines genetically engineered to express high or low levels of β2SP. Results: Increased levels of Notch signaling Hes1 and Jagged1 occurred in EA tissues and cell lines, compared to normal tissues. Loss of β2SP in MEFs cells increased Hes1 expression by 40 fold. In addition, SOX9, a target gene of Notch signaling and a documented stem cell marker was highly up-regulated in EA tumor cells and tissues. Expression of SOX9 was increased in the absence of β2SP in MEFs cells by 9 fold. Down-regulation of β2SP in SKTG-4, FLO-1 and BE3 EA cells by lentivirus shRNA led to increased SOX9 expression and enhanced nuclear localization of both active intracellular Notch1 domain (ICN1) and SOX9. Reintroducing β2SP into EA cells with knock down β2SP decreased SOX9 promoter activity by 10 fold. Concomitantly, an increased proportion of stem cells in β2SP knock down cells were identified using stem cell marker OCT3/4, indicating expansion of putative cancer stem cells in the absence of β2SP. Most interestingly, we observed a direct interaction between Smad3 and ICN1 via Smad3 MH1 domain by GST-pull down assays and that loss of β2SP increases the binding of Smad3 with ICN1 and induces Notch targets SOX9 and C-MYC transcription and decreased expression of its own targets P21, P27 and E-Cadherin. Conclusions: Our findings suggests that loss of TGF-β signaling adaptor β2SP may switch Smad3 function from tumor suppression to tumor promotion by binding intracellular Notch1 domain (ICN1) and activating Notch signaling. Thereby, a potential therapeutic value for targeted therapy in EA is in the setting of loss of β2SP/TGF-β with active Notch signaling.

Abstract 3243: Dysfunctional of TGF-β signaling via loss of β2SP activates SOX9 and C-MYC via Notch signaling in Barrett's esophageal adenocarcinoma

Abstract Background: Esophageal adenocarcinoma (EA) is often considered to arise from a clonal stem like population of cells, potentially responsible for its poor prognosis. TGF-β and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. We have demonstrated loss of TGF-β signaling components and activation of Notch signaling in Barrett's esophageal adenocarcinoma; however, their contributions to EA and the mechanisms of their action remain unclear. Methods: Immunoblotting and immunofluorescence were used to evaluate protein expression and localization. Notch targets Hes-1, SOX9 and C-MYC transcription were assayed using their luciferase reporters and Q-PCR. Functional studies were performed in β2SP wild type and mutant MEFs cells and in EA cell lines genetically engineered to express high or low levels of β2SP. Results: Increased levels of Notch signaling Hes1 and Jagged1 occurred in EA tissues and cell lines, compared to normal tissues. Loss of β2SP in MEFs cells increased Hes1 expression by 40 fold. In addition, SOX9, a target gene of Notch signaling and a documented stem cell marker was highly up-regulated in EA tumor cells and tissues. Expression of SOX9 was increased in the absence of β2SP in MEFs cells by 9 fold. Down-regulation of β2SP in SKTG-4, FLO-1 and BE3 EA cells by lentivirus shRNA led to increased SOX9 expression and enhanced nuclear localization of both active intracellular Notch1 domain (ICN1) and SOX9. Reintroducing β2SP into EA cells with knock down β2SP decreased SOX9 promoter activity by 10 fold. Concomitantly, an increased proportion of stem cells in β2SP knock down cells were identified using stem cell marker OCT3/4, indicating expansion of putative cancer stem cells in the absence of β2SP. Most interestingly, we observed a direct interaction between Smad3 and ICN1 via Smad3 MH1 domain by GST-pull down assays and that loss of β2SP increases the binding of Smad3 with ICN1 and induces Notch targets SOX9 and C-MYC transcription. Conclusions: Our findings suggests that loss of TGF-β signaling adaptor β2SP may switch Smad3 function from tumor suppression to tumor promotion by binding intracellular Notch1 domain (ICN1) and activating Notch signaling. Thereby, a potential therapeutic value for targeted therapy in EA is in the setting of loss of β2SP/TGF-β with active Notch signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3243. doi:1538-7445.AM2012-3243

Notch Signaling Modulates Hypoxia-Induced Neuroendocrine Differentiation of Human Prostate Cancer Cells

Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation (NED) has been associated with tumor progression, poor prognosis, and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavorable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells in vitro. Exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A, and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent downregulation of Notch-mediated signaling, as shown by reduced levels of the Notch target genes, Hes1 and Hey1. NED was promoted by attenuation of Hes1 transcription, as cells expressing a dominant-negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia downregulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen-independent cell lines, PC-3 and Du145, it did not change the extent of NED in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. Hypoxia induces NED of LNCaP cells in vitro, which seems to be driven by the inhibition of Notch signaling with subsequent downregulation of Hes1 transcription.

The Notch Signaling Pathway as a Suppressor of Myeloid Transformation

Abstract SCI-13Notch signaling is a central regulator of differentiation in a variety of organisms and tissue types. Its activity is controlled by the multi-subunit γ-secretase complex (γSE) complex. Although Notch signaling can play both oncogenic and tumor suppressor roles in solid tumors, in the hematopoietic system, it is exclusively oncogenic, notably in T cell acute lymphoblastic leukemia (T-ALL), a disease characterized by Notch1 activating mutations. We identified somatic inactivating Notch pathway mutations in a fraction of chronic myelomonocytic leukemia (CMML) patients. Inactivation of Notch signaling in mouse hematopoietic stem cells (HSC) results in an aberrant accumulation of granulocyte/monocyte progenitors (GMP), extramedullary hematopoieisis and the induction of CMML-like disease. Transcriptome analysis reveals that Notch signaling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. These studies identify a novel role for Notch signaling during early hematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumor-promoting and suppressive roles within the same tissue. These observations also suggest that Notch activity is not simply a promoter of the T cell lineage in the thymus but that Notch signaling thresholds could regulate commitment and/or survival of distinct hematopoietic lineages in the bone marrow. To address these issues in vivo, we have generated Notch receptor lineage tracing and activity reporter genetic tools. Analysis of these animal models identified unique novel functions for the Notch pathway during early bone marrow hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Kicking It Up and Down a Notch: An Important Switch in Pituitary Development and Disease.

The synthesis and release of the gonadotropins LH and FSH from the anterior pituitary are essential for fertility in males and females. Many cases of infertility that present with altered gonadotropin levels are idiopathic, making the identification of genes controlling gonadotrope development and function critically important. Notch signaling is a cell-cell communication pathway that has roles in precursor maintenance and cell fate assignment. The Notch targets HES1 and PROP1 are necessary to prevent premature cell specification in the developing pituitary gland and to produce a pituitary with the proper number of endocrine cells. We have determined that in addition to its role in organogenesis, Notch signaling must be suppressed for timely hormone production in gonadotropes. A candidate for suppressing Notch activity is NUMB, which causes degradation of the Notch receptor via interactions with the ubiquitin ligase Itch. NUMB is present in the developing mouse pituitary and is preferentially localized in gonadotropes in the anterior lobe of the adult. To determine the function of NUMB in the gonadotropes, Cre recombinase, driven by the αGSU promoter, was used to conditionally delete Numb and its homolog Numblike (cDKO). Female and male cDKO mice display signs of prematurely entering puberty, although Lhb and Fshb mRNA levels are unaffected. Both males and females are fertile, however female cDKO mice produce litters with a significantly fewer pups than control females or females bred to cDKO males. This study suggests Numb and Numblike may have a previously unidentified critical role in pituitary mediated reproduction. (platform)

A novel tumour-suppressor function for the Notch pathway in myeloid leukaemia

Notch signaling is a central regulator of differentiation in a variety of organisms and tissue types1. Its activity is controlled by the multi-subunit γ–secretase complex (γSE) complex2. Although Notch signaling can play both oncogenic and tumor suppressor roles in solid tumors, in the hematopoietic system, it is exclusively oncogenic, notably in T cell acute lymphoblastic leukemia (T-ALL), a disease characterized by Notch1 activating mutations3. Here we identify novel somatic inactivating Notch pathway mutations in a fraction of chronic myelomonocytic leukemia (CMML) patients. Inactivation of Notch signaling in mouse hematopoietic stem cells (HSC) resulted in an aberrant accumulation of granulocyte/monocyte progenitors (GMP), extramedullary hematopoieisis and the induction of CMML-like disease. Transcriptome analysis revealed that Notch signaling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. Our studies identify a novel role for Notch signaling during early hematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumor-promoting and suppressive roles within the same tissue.

Abstract 1765: Inhibition of Notch signaling by a Notch1 monoclonal antibody induces robust anti-tumor efficacy in T-cell acute lymphoblastic leukemia and breast cancer

Abstract Notch signaling is deregulated in T-cell acute lymphoblastic leukemia (T-ALL) and advanced solid tumors, making it an attractive target for oncology drug development. In this present report, we describe a novel mouse monoclonal antibody, Notch1 mAb, that specifically binds to the negative regulatory region (NRR) of human Notch1 receptor. Using a T-ALL cell line, HPB-ALL, that harbors mutations in Notch1 heterodimerization and PEST domains, we demonstrated that Notch1 mAb blocked Notch signaling by reduction of Notch1 intracellular domain (NICD) and down-regulation of Notch target genes, Hes-1 and cMyc. Notch1 mAb caused cell growth inhibition of HPB-ALL and several other T-ALL cell lines, via induction of cell cycle arrest and apoptosis. Notch1 mAb treatment resulted in robust NICD reduction and marked antitumor efficacy in HBP-ALL xenograft model. Additional mechanism-of-action studies revealed inhibition of tumor cell proliferation and induction of apoptosis in HPB-ALL tumors, suggesting that the anti-tumor activity of Notch1 mAb may be mediated by its direct effects on tumor cell growth or survival. Furthermore, this antibody led to a significant reduction in leukemia progenitor cells (LPCs) baring NOTCH1 mutation in bioluminescent humanized T-ALL LPC mouse models. In addition to its inhibitory effect on mutant Notch1, Notch1 mAb also displayed robust neutralization activity on wild type Notch1 and caused tumor growth inhibition in breast cancer models MDA-MB-231 and MX-1 by targeting the wild type receptor in these tumor types. Interestingly, using a gamma secretase inhibitor PF-03084014, we showed that Notch1 mAb and PF-03084014 elicited similar degree of biology responses in HPB-ALL cells. Our results indicate that antibodies that bind to NRR can act as potent inhibitors of Notch1 signaling and provide opportunities for development of novel cancer therapeutics for T-ALL and solid tumors. 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 1765. doi:10.1158/1538-7445.AM2011-1765

Abstract 2833: Functional cross-talk between the Wnt and Notch pathways regulate hyperplasia and/or colitis in response to bacterial infection

Abstract Notch and Wnt/β-catenin pathways play essential roles in intestinal development, homeostasis and neoplasia. Citrobacter rodentium (CR) induces transmissible murine colonic hyperplasia (TMCH) and variable degrees of inflammation and necrosis depending upon the genetic background. In the current study, we investigated functional cross-talk between the Notch and Wnt pathways and how they regulate colonic crypt hyperplasia and/or colitis in response to CR infection. Infection of NIH:Swiss outbred mice with CR induced gross thickening and rigidity of the distal colon and crypt hyperplasia which peaked by day 12, persisted until day 27 before declining by day 34. Nuclear staining for Notch target Hes-1 increased at days 6-27 before declining at day 34. Acute (5 days) treatment with Notch blocker dibenzazepine (DBZ; @10μmol/Kg) beginning on the 1st day of CR infection followed by euthanasia at day 6 blocked both Notch and Wnt signaling and induced goblet cells hyperplasia; acute DBZ treatment beginning on the 7th day of CR infection followed by euthanasia at day 12 inhibited Notch signaling without blocking the components of the Wnt pathway: both β-catenin and its downstream target Jagged-1, a Notch ligand, increased significantly at day 12 leading to crypt hyperplasia. Chronic (10 days) DBZ treatment beginning on the 1st day of CR infection followed by euthanasia at day 12 blocked both Notch and Wnt signaling, increased epithelial permeability and induced colitis. To directly establish that the disruption of mucus gel layer overlying the gut epithelium by DBZ may be the cause of colitis in CR infected animals, we utilized mice lacking Core 3 beta1,3-N-acetylglucosaminyltransferase, an enzyme important in synthesis of Core 3-derived O-glycans, the primary component of the intestinal mucins. The Core-3−/− mice are susceptible to experimental triggers of colitis. Infection of Core-3−/− mice with CR destroyed the intestinal barrier and induced colitis which was exacerbated following DBZ treatment. The changes accrued were not due to loss of colonic stemness as DBZ did not alter putative stem cell marker DCAMKL-1 expression in either strain. Dietary bael extract (4%) and curcumin (4%) restored the Notch/Wnt cross-talk and promoted crypt regeneration/hyperplasia in CR-infected/DBZ-treated SW mice. Thus, 1. TMCH exhibits active Notch and Wnt signaling. 2. Blocking Notch signaling early induces goblet cells hyperplasia but no colitis. 3. Acute but late stage blocking of Notch signaling does not abrogate Wnt/β-catenin-induced crypt hyperplasia. 4. Chronic defect in Notch signaling also blocks Wnt pathway and induces colitis in response to CR infection due to loss of barrier function and lack of crypt regeneration despite lack of alterations in colonic stemness. Thus, both Wnt and Notch cross-talk and epithelial barrier regulate hyperplasia and/or colitis in response to CR infection. 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 2833. doi:10.1158/1538-7445.AM2011-2833

Genetic Evidence That Intestinal Notch Functions Vary Regionally and Operate through a Common Mechanism of Math1 Repression

Notch signaling is active in many sites, and its diverse activities must require tissue-specific intermediaries, which are largely unknown. In the intestinal epithelium, Notch promotes crypt cell proliferation and inhibits goblet cell differentiation. Pharmacologic studies suggest that the latter effect occurs through the transcription factor Math1/Atoh1, which specifies all intestinal secretory cells. We tested this hypothesis using mouse mutants. Genetic loss of the Notch effector RBP-Jκ alone increases all intestinal secretory lineages, with variation between proximal and distal gut segments. This secretory cell excess observed with RBP-Jκ loss was blocked in the absence of Math1 in RBP-Jκ(Fl/Fl);Math1(Fl/Fl);Villin-Cre((ER-T2)) mice. Loss of both factors also restored progenitor replication, proving that Math1 is epistatic to Notch signaling in both secretory cell differentiation and crypt cell proliferation. Investigating mechanisms downstream of Math1, we found that expression of the known Notch effector protein Hes1 was predictably lost in RBP-Jκ(-/-) mice but surprisingly recovered in RBP-Jκ;Math1 compound conditional mutants. Furthermore, the cell cycle inhibitors p27(Kip1) and p57(Kip2) were selectively overexpressed in duodenal and ileal crypts, respectively, in RBP-Jκ-deficient mice. Regional activation of these products was completely abrogated in the absence of Math1. Thus, all intestinal Notch effects channel through the tissue-restricted factor Math1, which promotes secretory differentiation and cell cycle exit by regionally distinct mechanisms. Our data further suggest that, besides transmitting Notch signals, the transcription factor Hes1 acts downstream of Math1 to regulate expression of cell cycle inhibitors and intestinal crypt cell replication.

The Notch Target Hes1 Directly Modulates Gli1 Expression and Hedgehog Signaling: A Potential Mechanism of Therapeutic Resistance

Multiple developmental pathways including Notch, Hedgehog, and Wnt are active in malignant brain tumors such as medulloblastoma and glioblastoma (GBM). This raises the possibility that tumors might compensate for therapy directed against one pathway by upregulating a different one. We investigated whether brain tumors show resistance to therapies against Notch, and whether targeting multiple pathways simultaneously would kill brain tumor cells more effectively than monotherapy. We used GBM neurosphere lines to investigate the effects of a gamma-secretase inhibitor (MRK-003) on tumor growth, and chromatin immunoprecipitation to study the regulation of other genes by Notch targets. We also evaluated the effect of combined therapy with a Hedgehog inhibitor (cyclopamine) in GBM and medulloblastoma lines, and in primary human GBM cultures. GBM cells are at least partially resistant to long-term MRK-003 treatment, despite ongoing Notch pathway suppression, and show concomitant upregulation of Wnt and Hedgehog activity. The Notch target Hes1, a repressive transcription factor, bound the Gli1 first intron, and may inhibit its expression. Similar results were observed in a melanoma-derived cell line. Targeting Notch and Hedgehog simultaneously induced apoptosis, decreased cell growth, and inhibited colony-forming ability more dramatically than monotherapy. Low-passage neurospheres isolated from freshly resected human GBMs were also highly susceptible to coinhibition of the two pathways, indicating that targeting multiple developmental pathways can be more effective than monotherapy at eliminating GBM-derived cells. Notch may directly suppress Hedgehog via Hes1 mediated inhibition of Gli1 transcription, and targeting both pathways simultaneously may be more effective at eliminating GBMs cells.

Rabconnectin-3 Is a Functional Regulator of Mammalian Notch Signaling

The Notch signaling pathway is important for cell fate decisions in embryonic development and adult life. Defining the functional importance of the Notch pathway in these contexts requires the elucidation of essential signal transduction components that have not been fully characterized. Here, we show that Rabconnectin-3B is required for the Notch pathway in mammalian cells. siRNA-mediated silencing of Rabconnectin-3B in mammalian cells attenuated Notch signaling and disrupted the activation and nuclear accumulation of the Notch target Hes1. Rabconnectin-3B knockdown also disrupted V-ATPase activity in mammalian cells, consistent with previous observations in Drosophila. Pharmacological inhibition of the V-ATPase complex significantly reduced Notch signaling in mammalian cells. Finally, Rabconnectin-3B knockdown phenocopied functional disruption of Notch signaling during osteoclast differentiation. Collectively, these findings define an important role for Rabconnectin-3 and V-ATPase activity in the Notch signaling pathway in mammalian cells.

Preclinical analyses of a new gamma-secretase inhibitor targeting notch signaling in melanoma.

8546 Background: Treatment of metastatic melanoma remains a challenge because most cases are refractory to chemotherapy. The lack of effective treatment and the propensity for rapid disease progression warrant further investigations into cellular pathways that may provide melanoma cells with a survival benefit. Several reports have demonstrated a role for aberrant Notch signaling in melanoma genesis or progression, prompting us to explore if targeting this pathway is a valid therapeutic approach against melanoma. Methods: To investigate the potential benefits of Notch inhibition in melanoma, we are using RO4929097, a novel inhibitor of gamma secretase, a key component of the enzymatic complex that cleaves and activates Notch. RO4929097 is completing phase I dose escalation in cancer patients and a CTEP-sponsored phase II clinical trial in melanoma is currently under review. We assessed the effects of RO4929097 on human melanoma cell lines both in vitro (proliferation assays, colony formation assay, sphere formation assay) and in vivo (xenograft tumor growth). Results: Our in vitro assays revealed a dose-dependent effect of RO4929097 on the levels of Notch targets Hes1 and Hey1, measured by qPCR. Moreover, sensitive cells displayed phenotypic and functional changes in response to treatment, including reduced proliferation and impaired ability to organize in tridimensional spheroids and to form colonies in soft agar. In vivo studies using human melanoma xenografts in NOD/SCID mice showed delayed tumor growth following daily RO4929097 oral administration at 10 mg/kg of mouse. Tumors developed by RO4929097-treated mice displayed extensive necrosis and significantly lower proliferative index. Strikingly, RO4929097 administration interfered also with the growth of preexisting tumors, blunting their invasive potential. As opposed to other gamma-secretase inhibitors, RO4929097 did not produce overt toxicity. Conclusions: Our data support Notch inhibition as a targeted therapy against melanoma. Ongoing studies aimed at identifying the molecular bases of response or resistance to RO4929097 might be used for patient selection in future clinical trials. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Roche Roche

Abstract 3905: Structure–function relationships between RUNX1-MTG fusion proteins and core components of the Notch transcription complex

Abstract The myeloid translocation genes (MTG) are preferred targets of chromosomal translocations with the Runx1 gene in acute myeloid leukemia (AML) pathogenesis. The resulting fusion proteins, RUNX1-MTG8 and RUNX1-MTG16, influence gene expression programs that control self-renewal and differentiation in hematopoiesis. How they engage the machinery of self-renewal to alter hematopoietic development is poorly understood. To gain additional insights into this question, we investigated structure–function relationships between RUNX1-MTG fusion proteins and core components of the Notch transcription complex, a critical determinant of cell fate specification in diverse tissues. The Notch transcription complex is composed minimally of the transcriptional repressor CSL, the intracellular domain of Notch receptors (N-ICD) and the transcriptional co-activator Mastermind (MAML). We show that RUNX1-MTG fusion proteins transactivate a Notch-responsive element from the Hes1 promoter. Using MTG16 as a model protein, we show that N-ICDs interact with a discrete domain in the MTG N-terminus. We translated these findings to the fusion proteins and found N-ICD binding was retained by RUNX1-MTGs and a RUNX1-MTG16 mutant that lacks the N1-ICD binding site. In parallel experiments, we defined the binding region for MTG proteins and their fusion protein derivatives on the N1-ICD. Informed by these structural relationships, we utilized RUNX1-MTG proteins or mutants that lack N1-ICD binding sites in transcriptional reporter assays. Our findings suggest that RUNX1-MTG fusion proteins alter transcription of the Notch target gene, Hes1 and intimate a structure–function relationship between RUNX1-MTG fusion proteins and Notch signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3905.

Notch ligand-dependent gene expression in human endometrial stromal cells

The purpose of this study is to investigate the expression patterns and role of Notch signaling in human endometrial cells. Notch receptors, Notch 1–3 were expressed in both endometrial epithelial and stromal cells. Notch ligands, Jag1 and Dll4 and Notch target genes, Hes1 and Hey1 were predominantly expressed in endometrial epithelial cells and scarce in stromal cells. Increased de novo synthesis of Dll4 or Jag1 in stromal cells by retroviral delivery significantly induced Hes1 and Hey1. Evaluations of global gene expression by microarrays revealed that more than 400 genes in stromal cells were significantly regulated by Jag1. Gene annotation-based functional analysis classified these genes into biological processes of cell adhesion, cell structure and motility, cell communication, cell cycle, and angiogenesis. This study provides evidence that Notch ligands control the Notch gene activities and may enhance development of human endometrium.

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 ( Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.

Defects in Notch1 upregulation upon activation of T Cells from patients with systemic lupus erythematosus are related to lupus disease activity

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of autoantibodies and deposition of immune complexes in various organs. T cells play a central role in driving disease progression, and multiple defects in T cells from patients with SLE have been uncovered. Notch signalling is an evolutionarily well-conserved signalling cascade involved in the proliferation, differentiation and apoptosis of T lymphocytes during development and peripheral effector functions. In this study, we investigated the correlation between expression of Notch receptor and the severity of SLE disease. On the contrary to T lymphocytes from healthy controls (n=11), Tlymphocytes from patients with active SLE (n=12) failed to upregulate Notch1 upon in-vitro stimulation as quantified by quantitative real time RT-PCR (P<or=0.025). Among patients with inactive SLE (n=10), those with late onset of flare exhibited significantly less Notch1 upregulation compared with SLE patients with remission. Expression of the Notch target genes, Hes1 and deltex, was also lower in patients with active SLE. The decrease in Notch1 mRNA expression was consistent with less Notch1 protein expression in patients with active SLE. The defects in Notch1 upregulation correlated with decreased proliferation, CD25 and Foxp3 expression upon stimulation in vitro. Taken together, the failure of T cells to upregulate Notch1 upon activation may be a key feature of active SLE and a potential therapeutic target.

Cooperation of Gata3, c-Myc and Notch in malignant transformation of double positive thymocytes

Gata transcription factors are critical regulators of proliferation and differentiation implicated in various human cancers, but specific genes activated by Gata proteins remain to be identified. We previously reported that enforced expression of Gata3 during T cell development in CD2-Gata3 transgenic mice induced CD4 +CD8 + double-positive (DP) T cell lymphoma. Here, we show that the presence of the DO11.10 T-cell receptor transgene, which directs DP cells towards the CD4 lineage, resulted in enhanced lymphoma development and a dramatic increase in thymocyte cell size in CD2-Gata3 transgenic mice. CD2-Gata3 DP cells expressed high levels of the proto-oncogene c-Myc but the Notch1 signaling pathway, which is known to induce c-Myc, was not activated. Gene expression profiling showed that in CD2-Gata3 lymphoma cells transcription of c-Myc and its target genes was further increased. A substantial fraction of CD2-Gata3 lymphomas had trisomy of chromosome 15, leading to an increased c-Myc gene dose. Interestingly, most lymphomas showed high expression of the Notch targets Deltex1 and Hes1, often due to activating Notch1 PEST domain mutations. Therefore, we conclude that enforced Gata3 expression converts DP thymocytes into a pre-malignant state, characterized by high c-Myc expression, whereby subsequent induction of Notch1 signaling cooperates to establish malignant transformation. The finding that Gata3 regulates c-Myc expression levels, in a direct or indirect fashion, may explain the parallel phenotypes of mice with overexpression or deficiency of either of the two transcription factors.