Pharmacological Inhibition Of Notch Signaling Research Articles

O-189 Extracellular vesicles derived from human myometrial cells support endometrial mesenchymal stromal/stem cells

Abstract Study question Whether other forms of intercellular communication such as extracellular vesicles (EVs) participate in stem cell maintenance in the human endometrium. Summary answer The present study revealed the involvement of EVs as a novel intercellular communication method between endometrial mesenchymal stem cells (eMSC) and their surrounding niche. What is known already In human endometrium, many studies have demonstrated the importance of EVs in mediating various physiological as well as pathological processes. Our group has demonstrated that the myometrial cells are candidate niche cells of eMSC modulating their biological function. We also demonstrate the Notch signaling pathway is involved in endometrial stem cell regulation. Although classical Notch signaling relies on direct cell contact dependent interactions, this pathway can also be activated at a distance by EVs containing Notch ligands. Therefore, we hypothesized that certain Notch ligand(s) are packaged into the myometrial EVs to mediate stem cell functions. Study design, size, duration Sequential beading with magnetic beads coated with anti-CD140b and anti-CD146 antibodies was used to isolate eMSC (CD140b+CD146+ cells) from endometrial tissues from proliferative (n = 15) and secretory (n = 10) phase. Myometrial derived EVs (n = 17) were isolated by ultracentrifugation. The EVs were characterized by transmission electron microscopy and western blotting. Participants/materials, setting, methods Endometrial samples obtained from women aged 41 – 52 years undergoing total abdominal hysterectomy. EMSC were cocultured with myometrial EVs and the percentage of eMSC was analysed by flow cytometry. Blockage the secretion of EVs was performed by transfection of Rab27a siRNA. Western blot analysis and gene silencing approach validated the role of Notch signaling in eMSC. The therapeutic features of transplanted eMSC/myometrial EVs was determined using the mouse injured endometrial model. Main results and the role of chance EVs released from myometrial cells can be internalized by eMSC, leading to a significant effect on stemness of eMSC. Pharmacological inhibition of Notch signaling with DAPT or silencing of Notch 1 nullified these stimulatory effects. Myometrial EVs contains a high amount of the notch ligand – JAG1, thus inducing a strong Notch activity in eMSC. When JAG1 was silenced in the myometrial EVs, the self-renewal and clonogenic activity was reduced. Combined transplantation of eMSC with myometrial EVs improves the therapeutic effect of eMSC in endometrial regeneration in vivo. The observed therapeutic feature was potentially achieved by elevating the cell proliferation and suppressing apoptosis in the injured mouse endometrium. Limitations, reasons for caution Whether other signaling pathway are involved in the stimulatory effect of myometrial EVs in eMSC activities remains unknown. Also, EVs derived from other endometrial cell types i.e. epithelial cells may also regulate eMSC function. Wider implications of the findings Our findings revealed that myometrial EVs stimulated the biological function of eMSC via the Notch signaling. In the near future, it would be important to explore whether Notch signaling pathway plays a role in the injured mouse endometrium transplanted with eMSC/myometrial EVs. Trial registration number Not applicable

Endothelial cell senescence exacerbates pulmonary hypertension by inducing juxtacrine Notch signaling in smooth muscle cells

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by a progressive increase in pulmonary artery pressure caused by pathological pulmonary artery remodeling. Here, we demonstrate that endothelial cell (EC) senescence plays a negative role in pulmonary hypertension via juxtacrine interaction with smooth muscle cells (SMCs). By using EC-specific progeroid mice, we discovered that EC progeria deteriorated vascular remodeling in the lungs, and exacerbated pulmonary hypertension in mice. Mechanistically, senescent ECs overexpressed Notch ligands, which resulted in increased Notch signaling and activated proliferation and migration capacities in neighboring SMCs. Pharmacological inhibition of Notch signaling reduced the effects of senescent ECs on SMCs functions invitro, and improved the worsened pulmonary hypertension in EC-specific progeroid mice invivo. Our findings show that EC senescence is a critical disease-modifying factor in PAH and that EC-mediated Notch signaling is a pharmacotherapeutic target for the treatment of PAH, particularly in the elderly.

BCL6 and the Notch pathway: a signaling axis leading to a novel druggable biotarget in triple negative breast cancer.

The transcriptional repressor B-cell lymphoma 6 (BCL6) is dysregulated in several neoplasms, but its role in triple negative breast cancer (TNBC), a highly aggressive subtype which lacks effective treatment, is unclear. The presence of intratumoral cancer stem cells (CSCs) is a main cause of tumor relapse. The Notch signaling pathway is crucial for regulating CSC self-renewal and promoting breast cancer (BC) development and resistance to anticancer therapies. Here, we investigated signaling cascades of BCL6 in the CSC compartment of TNBCs, and the mechanisms that govern its activity, mainly through Notch signaling. Gene expression, somatic copy number alterations and clinical data from the Cancer Genome Atlas and METABRIC were accessed through theXena and cbioportal browsers. Public transcriptome profiles from TNBC datasets were retrieved from theGene Expression Omnibus. Mammosphere formation efficiency was calculated after BCL6 knockdown via transient siRNAtransfection, stable silencing or pharmacological inhibition. The effects exhibited via BCL6 inhibition in putative TNBC stem-like cells were evaluated by immunofluorescence and qRT-PCR analyses. Chromatin immunoprecipitation experiments were performed to validate a putative BCL6 responsive element located in the first intron of the Numb gene and to define the circuit of corepressors engaged by BCL6 following its inhibition. Immunoprecipitation assays were carried out to investigate a novel interaction at the basis of BCL6 control of CSC activity in TNBC. In silico analyses of benchmarked public datasets revealed a significant enrichment of BCL6 in cancer stemness related pathways, particularly of Notch signaling in TNBC. In vitro stable inhibition of BCL6 significantly reduced tumor cell growth and, accordingly, we found thatthe mammosphere formation efficiency of BCL6 silencedcells was significantly impaired by pharmacological inhibition of Notch signaling. BCL6 was found to be expressed at significantly higher levels in TNBC mammospheres than in their adherent counterparts, and loss of BCL6 function significantly decreased mammosphere formation with preferential targeting of CD44-positive versus ALDH-positive stem-like cells. Functional interplay between BCL6 and the chromatin remodeling factor EZH2 triggered the BCL6/Notch stemness signaling axis via inhibition of Numb transcription. Our results may be instrumental for the prospective design of combination treatment strategies that selectively target novel TNBC-associated biomarker(s) whose activity is implicated in the regulation of cancer stemness (such as BCL6) and molecules in developmentally conserved signaling pathways (such as Notch) to achieve long-lasting tumor control and improve patient outcomes.

Notch signaling enhances bone regeneration in the zebrafish mandible.

Loss or damage to the mandible caused by trauma, treatment of oral malignancies, and other diseases is treated using bone-grafting techniques that suffer from numerous shortcomings and contraindications. Zebrafish naturally heal large injuries to mandibular bone, offering an opportunity to understand how to boost intrinsic healing potential. Using a novel her6:mCherry Notch reporter, we show that canonical Notch signaling is induced during the initial stages of cartilage callus formation in both mesenchymal cells and chondrocytes following surgical mandibulectomy. We also show that modulation of Notch signaling during the initial post-operative period results in lasting changes to regenerate bone quantity one month later. Pharmacological inhibition of Notch signaling reduces the size of the cartilage callus and delays its conversion into bone, resulting in non-union. Conversely, conditional transgenic activation of Notch signaling accelerates conversion of the cartilage callus into bone, improving bone healing. Given the conserved functions of this pathway in bone repair across vertebrates, we propose that targeted activation of Notch signaling during the early phases of bone healing in mammals may both augment the size of the initial callus and boost its ossification into reparative bone.

Notch-triggered maladaptation of liver sinusoidal endothelium aggravates nonalcoholic steatohepatitis through endothelial nitric oxide synthase.

Although NASH can lead to severe clinical consequences, including cirrhosis and hepatocellular carcinoma, no effective treatment is currently available for this disease. Increasing evidence indicates that LSECs play a critical role in NASH pathogenesis; however, the mechanisms involved in LSEC-mediated NASH remain to be fully elucidated. In the current study, we found that LSEC homeostasis was disrupted and LSEC-specific gene profiles were altered in methionine-choline-deficient (MCD) diet-induced NASH mouse models. Importantly, Notch signaling was found to be activated in LSECs of NASH mice. To then investigate the role of endothelial Notch in NASH progression, we generated mouse lines with endothelial-specific Notch intracellular domain (NICD) overexpression or RBP-J knockout to respectively activate or inhibit Notch signaling in endothelial cells. Notably, endothelial-specific overexpression of the NICD accelerated LSEC maladaptation and aggravated NASH, whereas endothelial cell-specific inhibition of Notch signaling restored LSEC homeostasis and improved NASH phenotypes. Furthermore, we demonstrated that endothelial-specific Notch activation exacerbated NASH by inhibiting endothelial nitric oxide synthase (eNOS) transcription, whereas administration of the pharmacological eNOS activator YC-1 alleviated hepatic steatosis and lipid accumulation resulting from Notch activation. Finally, to explore the therapeutic potential of using Notch inhibitors in NASH treatment, we applied two gamma-secretase inhibitors-DAPT and LY3039478-in an MCD diet-induced mouse model of NASH, and found that both inhibitors effectively ameliorated hepatic steatosis, inflammation, and liver fibrosis. Endothelial-specific Notch activation triggered LSEC maladaptation and exacerbated NASH phenotypes in an eNOS-dependent manner. Genetic and pharmacological inhibition of Notch signaling effectively restored LSEC homeostasis and ameliorated NASH progression.

Activation of Notch and Myc Signaling via B-cell-Restricted Depletion of Dnmt3a Generates a Consistent Murine Model of Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc-expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. SIGNIFICANCE: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.

Abstract 12266: Endothelial Cells Senescence Exacerbates Pulmonary Hypertension Through Notch-Induced Pulmonary Arterial Smooth Muscle Cells Proliferation and Migration

Background: Endothelial cell (EC) dysfunction is critically involved in the pathogenesis of pulmonary arterial hypertension (PAH) by mediating structural changes in vasculatures. Various stress that promotes EC dysfunction also induces premature senescence in EC; however, the role of EC senescence in the development of PAH remains unknown. Hypothesis: We hypothesized that senescent ECs play a significant role in the development of PAH. Methods: We generated EC-specific progeroid mice that overexpress the dominant-negative form of telomere repeat binding factor 2 under the control of the VE-cadherin promoter. Following chronic hypoxia exposure, PH phenotypes were assessed by RVSP and lung histology. Gene expression was assessed by RT-qPCR. The interaction of pulmonary artery ECs (PAECs) and pulmonary artery smooth muscle cells (PASMCs) was analyzed using indirect and direct co-culture systems. In addition, gamma-secretase inhibitor (DAPT) was used to inhibit Notch signaling both in the in-vitro and in-vivo studies. Results: The transgene expression was specifically expressed and fairly distributed in various pulmonary blood vessels in EC-specific progeroid mice. After three weeks of hypoxia exposure, EC-specific progeroid mice exhibited exacerbated pulmonary hypertension (PH) phenotype, accompanied by the prominent medial thickening and enhanced proliferation capacity of SMCs in the distal pulmonary arteries. Interestingly, contact-mediated interaction with senescent ECs increased proliferation and migration capacities in SMCs, while the absence of EC-SMC contact abolished those effects. Mechanistically, senescent ECs expressed higher Notch ligands than in young ECs, leading to the enhanced Notch signaling in PASMCs. Consistently, pharmacological inhibition of Notch signaling attenuated senescent ECs’ effects on SMCs in vitro and the PH phenotype in EC-specific progeroid mice in vivo. Conclusions: These findings established the critical roles of senescent ECs in the PAH pathogenesis, suggesting a novel pathological-targeted therapeutic approach for PAH.

Bone-Targeted Pharmacological Inhibition of Notch Signaling As a Novel Approach to Inhibit Myeloma Growth and Bone Destruction

Notch signaling between myeloma cells and cells of the bone marrow microenvironment favors growth and survival of myeloma cells and increases osteoclast formation. In vitro and in vivo studies demonstrated that systemic inhibition of Notch signaling using gamma-secretase inhibitors (GSIs) decreases myeloma cell growth and reduces osteoclast generation. However, the use of GSIs in the clinic is limited by the ocurrence of severe adverse side effects as fatigue, skin disorders, and acute gastrointestinal toxicity. To circumvent the side effects associated with systemic inhibition of Notch signaling using GSIs, we synthesized a novel Notch inhibitor by linking GSI-XII to an inactive bone-targeting molecule (BT). BT-GSI was designed to direct the conjugate to bone where the linker is cleaved under the acidic environment generated by osteoclast activity, thus locally releasing the GSI.In vitro, the control unconjugated GSI decreased Notch target gene expression (Hes1) in 5TGM1 myeloma cells, but BT-GSI had no effect. However, when both GSI and BT-GSI were pre-incubated at low pH to mimic the acidic conditions in resorption sites, equal inhibition of Notch target gene expression was observed. Ex vivo, both GSI and BT-GSI (non-pre-incubated) similarly decreased Hes1/5 expression in whole bone organ cultures that reproduce conditions present in the bone microenvironment. In vivo, administration of BT-GSI (5µg/g, 3xwk) to normal 4-month old female mice for 2 weeks was well tolerated and no skin issues were observed. Mice treated with BT-GSI exhibited decreased Hey2, Hes5, and Hes7 mRNA expression in whole bone preparations, but not in brain or gut, compared to vehicle-treated mice. Further, BT-GSI did not increase Apsidin expression in the gut, a biomarker of gastrointestinal toxicity. BT-GSI-treated mice exhibited decreased serum levels of CTX (-40%), a bone resorption marker, and upregulated Opg mRNA expression in bone, thereby decreasing the Rankl/Opg ratio. Consistent with these findings, BT-GSI-treated mice exhibited a 50% decreased in the bone surface covered by osteoclasts compared to vehicle-treated control mice. BT-GSI treated mice exhibited higher total (+3%), femoral (+4%), and spinal (+7%) BMD compared to control mice. Further, mice receiving BT-GSI had increased cancellous bone volume (+25%) and trabecular thickness (+10%) compared to vehicle-treated mice, as quantified by microCT. Serum levels of the bone formation marker P1NP, as well as bone formation and mineral apposition rates, number of osteoblasts, and the expression of osteoblast markers, including Wnt target genes and the osteocyte- derived Sost/Sclerostin remained unchanged in bones of BT-GSI-treated mice compared to control mice.Taken together, these findings demonstrate that BT-GSI induces bone specific Notch inhibition, reduces osteoclast formation without affecting osteoblast activity, favoring bone gain, and lacks gut toxicity. Thus, BT-GSI is a promising approach to inhibit the growth of myeloma cells and prevent bone loss in myeloma patients, while circumventing the deleterious side effects of this class of inhibitors in other tissues. DisclosuresDelgado-Calle:PharmaMar: Research Funding. Roodman:PharmaMar: Research Funding. Bellido:PharmaMar: Research Funding.

In Vitro Evaluation of Clinical Candidates of γ-Secretase Inhibitors: Effects on Notch Inhibition and Promoting Beige Adipogenesis and Mitochondrial Biogenesis.

Inhibition of Notch signaling has been recently demonstrated to promote beige adipocyte biogenesis. However, most γ-secretase inhibitors (GSIs) used to achieve pharmacological inhibition of Notch signaling are at the basic research or preclinical stage, limiting the translation of fundamental findings into clinical practice. This present study aimed to evaluate the potential of several clinical candidates of GSIs as browning agents for the treatment of obesity. Seven GSIs that are clinical candidates for the treatment of Alzheimer's disease or cancer were selected and their impacts on Notch inhibition as well as promoting beige biogenesis were compared using in vitro culture of 3T3-L1 preadipocytes. Four compounds(i.e.RO4929097, PF-03084014, LY3039478, and BMS-906024) that efficiently inhibited the expression of Notch target genes in 3T3-L1 preadipocytes were identified. Moreover, these compounds were optimized for dose-dependent effects at three gradient concentrations (0.5, 1, and 10μM) to promote beige adipogenesis and mitochondrial biogenesis in 3T3-L1 preadipocytes without causing severe cytotoxicity. Our findings not only highlight the potential of cross-therapeutic application of these GSIs for obesity treatment via inhibition of γ-secretase-mediated processing of Notch signaling, but also provide important experimental evidence to support further design and development of clinically translatable Notch-inhibiting drug delivery systems.

Activation of Notch1 signaling in podocytes by glucose-derived AGEs contributes to proteinuria

IntroductionAdvanced glycation end-products (AGEs) are implicated in the pathogenesis of diabetic nephropathy (DN). Previous studies have shown that AGEs contribute to glomerulosclerosis and proteinuria. Podocytes, terminally differentiated epithelial cells of...

Pharmacological inhibition of Notch signaling regresses pre-established abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is characterized by transmural infiltration of myeloid cells at the vascular injury site. Previously, we reported preventive effects of Notch deficiency on the development of AAA by reduction of infiltrating myeloid cells. In this study, we examined if Notch inhibition attenuates the progression of pre-established AAA and potential implications. Pharmacological Notch inhibitor (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester; DAPT) was administered subcutaneously three times a week starting at day 28 of angiotensin II (AngII) infusion. Progressive increase in pulse wave velocity (PWV), maximal intra-luminal diameter (MILD) and maximal external aortic diameter (MEAD) were observed at day 56 of the AngII. DAPT prevented such increase in MILD, PWV and MEAD (P < 0.01). Histologically, the aortae of DAPT-treated Apoe−/− mice had significant reduction in inflammatory response and elastin fragmentation. Naked collagen microfibrils and weaker banded structure observed in the aortae of Apoe−/− mice in response to AngII, were substantially diminished by DAPT. A significant decrease in the proteolytic activity in the aneurysmal tissues and vascular smooth muscle cells (vSMCs) was observed with DAPT (P < 0.01). In human and mouse AAA tissues, increased immunoreactivity of activated Notch signaling correlated strongly with CD38 expression (R2 = 0.61). Collectively, we propose inhibition of Notch signaling as a potential therapeutic target for AAA progression.

Inhibition of Notch signaling rescues cardiovascular development in Kabuki Syndrome.

Kabuki Syndrome patients have a spectrum of congenital disorders, including congenital heart defects, the primary determinant of mortality. Seventy percent of Kabuki Syndrome patients have mutations in the histone methyl-transferase KMT2D. However, the underlying mechanisms that drive these congenital disorders are unknown. Here, we generated and characterized zebrafish kmt2d null mutants that recapitulate the cardinal phenotypic features of Kabuki Syndrome, including microcephaly, palate defects, abnormal ear development, and cardiac defects. The cardiac phenotype consists of a previously unknown vasculogenesis defect that affects endocardium patterning and, consequently, heart ventricle lumen formation. Additionally, zebrafish kmt2d null mutants have angiogenesis defects depicted by abnormal aortic arch development, hyperactive ectopic blood vessel sprouting, and aberrant patterning of the brain vascular plexus. We demonstrate that zebrafish kmt2d null mutants have robust Notch signaling hyperactivation in endocardial and endothelial cells, including increased protein levels of the Notch transcription factor Rbpj. Our zebrafish Kabuki Syndrome model reveals a regulatory link between the Notch pathway and Kmt2d during endothelium and endocardium patterning and shows that pharmacological inhibition of Notch signaling rebalances Rbpj protein levels and rescues the cardiovascular phenotype by enhancing endothelial and endocardial cell proliferation and stabilizing endocardial patterning. Taken together, these findings demonstrate that Kmt2d regulates vasculogenesis and angiogenesis, provide evidence for interactions between Kmt2d and Notch signaling in Kabuki Syndrome, and suggest future directions for clinical research.

Peroxiredoxin 6 Confers Protection Against Nonalcoholic Fatty Liver Disease Through Maintaining Mitochondrial Function.

Aims: Nonalcoholic fatty liver disease (NAFLD) is accompanied by excessive reactive oxygen species (ROS) production, which has been suggested in several studies to link with mitochondrial function. However, the mechanistic role of ROS-mediated regulation of mitochondrial function in NAFLD has not been elucidated. Since peroxiredoxin 6 (PRDX6) is the only member of the antioxidant PRDX family that translocates to damaged mitochondria, we investigated the PRDX6-mediated antisteatotic mechanism using genetically modified mice and cells. Results: PRDX6 mice were more protective to lipid accumulation, liver injury, and insulin resistance after a high-fat diet. Mechanistically, PRDX6 is required for induction of mitochondrial antioxidant action and beta-oxidation through maintaining mitochondrial integrity and subsequently prevents ROS-induced lipogenesis. Interestingly, oxidative stress-induced Notch signaling was suppressed in PRDX6 mice compared with wild-type mice, and genetic and pharmacological inhibition of Notch signaling improved lipid accumulation. Finally, PRDX knockdown or Notch inhibition reduced induction of mitophagy. PRDX6 antagonizes positive feedback loop between lipid accumulation and ROS production through regulation of mitochondrial function. Innovation: For the first time, we demonstrate that PRDX6 maintains mitochondria integrity under oxidative stress and protects against NAFLD progression by inhibition of Notch signaling. Conclusion: This study describes a novel molecular mechanism underlying the antisteatotic activity of PRDX6, which may be a new therapeutic strategy for the treatment of NAFLD.

Notch Signaling Mediates Astrocyte Abnormality in Spinal Muscular Atrophy Model Systems

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by the degeneration of spinal motor neurons and muscle atrophy. The disease is mainly caused by low level of the survival motor neuron (SMN) protein, which is coded by two genes, namely SMN1 and SMN2, but leads to selective spinal motor neuron degeneration when SMN1 gene is deleted or mutated. Previous reports have shown that SMN-protein-deficient astrocytes are abnormally abundant in the spinal cords of SMA model mice. However, the mechanism of the SMN- deficient astrocyte abnormality remains unclear. The purpose of this study is to identify the cellular signaling pathways associated with the SMN-deficient astrocyte abnormality and propose a candidate therapy tool that modulates signaling. In the present study, we found that the astrocyte density was increased around the central canal of the spinal cord in a mouse SMA model and we identified the dysregulation of Notch signaling which is a known mechanism that regulates astrocyte differentiation and proliferation, in the spinal cord in both early and late stages of SMA pathogenesis. Moreover, pharmacological inhibition of Notch signaling improved the motor functional deficits in SMA model mice. These findings indicate that dysregulated Notch signaling may be an underlying cause of SMA pathology.

Screen for modulators of atonal homolog 1 gene expression using notch pathway-relevant gene transcription based cellular assays.

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix 9 (bHLH) transcription factor acting downstream of Notch and is required for the differentiation of sensory hair cells in the inner ear and the specification of secretory cells during the intestinal crypt cell regeneration. Motivated by the observations that the upregulation of Atoh1 gene expression, through genetic manipulation or pharmacological inhibition of Notch signaling (e.g. γ-secretase inhibitors, GSIs), induces ectopic hair cell growth in the cochlea of the inner ear and partially restores hearing after injuries in experimental models, we decided to identify small molecule modulators of the Notch-Atoh1 pathway, which could potentially regenerate hair cells. However, the lack of cellular models of the inner ear has precluded the screening and characterization of such modulators. Here we report using a colon cancer cell line LS-174T, which displays Notch inhibition-dependent Atoh1 expression as a surrogate cellular model to screen for inducers of Atoh1 expression. We designed an Atoh1 promoter-driven luciferase assay to screen a target-annotated library of ~6000 compounds. We further developed a medium throughput, real-time quantitative RT-PCR assay measuring the endogenous Atoh1 gene expression to confirm the hits and eliminate false positives from the reporter-based screen. This strategy allowed us to successfully recover GSIs of known chemotypes. This LS-174T cell-based assay directly measures Atoh1 gene expression induced through Notch-Hes1 inhibition, and therefore offers an opportunity to identify novel cellular modulators along the Notch-Atoh1 pathway.

NOTCH3 expression is linked to breast cancer seeding and distant metastasis

BackgroundDevelopment of distant metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which includes dissemination of cancer cells from the primary tumor to secondary organs. NOTCH developmental signaling plays a critical role in promoting epithelial-to-mesenchymal transition, tumor stemness, and metastasis. Although all four NOTCH receptors show oncogenic properties, the unique role of each of these receptors in the sequential stepwise events that typify the invasion-metastasis cascade remains elusive.MethodsWe have established metastatic xenografts expressing high endogenous levels of NOTCH3 using estrogen receptor alpha-positive (ERα+) MCF-7 breast cancer cells with constitutive active Raf-1/mitogen-associated protein kinase (MAPK) signaling (vMCF-7Raf-1) and MDA-MB-231 triple-negative breast cancer (TNBC) cells. The critical role of NOTCH3 in inducing an invasive phenotype and poor outcome was corroborated in unique TNBC cells resulting from a patient-derived brain metastasis (TNBC-M25) and in publicly available claudin-low breast tumor specimens collected from participants in the Molecular Taxonomy of Breast Cancer International Consortium database.ResultsIn this study, we identified an association between NOTCH3 expression and development of metastases in ERα+ and TNBC models. ERα+ breast tumor xenografts with a constitutive active Raf-1/MAPK signaling developed spontaneous lung metastases through the clonal expansion of cancer cells expressing a NOTCH3 reprogramming network. Abrogation of NOTCH3 expression significantly reduced the self-renewal and invasive capacity of ex vivo breast cancer cells, restoring a luminal CD44low/CD24high/ERαhigh phenotype. Forced expression of the mitotic Aurora kinase A (AURKA), which promotes breast cancer metastases, failed to restore the invasive capacity of NOTCH3-null cells, demonstrating that NOTCH3 expression is required for an invasive phenotype. Likewise, pharmacologic inhibition of NOTCH signaling also impaired TNBC cell seeding and metastatic growth. Significantly, the role of aberrant NOTCH3 expression in promoting tumor self-renewal, invasiveness, and poor outcome was corroborated in unique TNBC cells from a patient-derived brain metastasis and in publicly available claudin-low breast tumor specimens.ConclusionsThese findings demonstrate the key role of NOTCH3 oncogenic signaling in the genesis of breast cancer metastasis and provide a compelling preclinical rationale for the design of novel therapeutic strategies that will selectively target NOTCH3 to halt metastatic seeding and to improve the clinical outcomes of patients with breast cancer.

JunB defines functional and structural integrity of the epidermo-pilosebaceous unit in the skin

Transcription factors ensure skin homeostasis via tight regulation of distinct resident stem cells. Here we report that JunB, a member of the AP-1 transcription factor family, regulates epidermal stem cells and sebaceous glands through balancing proliferation and differentiation of progenitors and by suppressing lineage infidelity. JunB deficiency in basal progenitors results in a dermatitis-like syndrome resembling seborrheic dermatitis harboring structurally and functionally impaired sebaceous glands with a globally altered lipid profile. A fate switch occurs in a subset of JunB deficient epidermal progenitors during wound healing resulting in de novo formation of sebaceous glands. Dysregulated Notch signaling is identified to be causal for this phenotype. In fact, pharmacological inhibition of Notch signaling can efficiently restore the lineage drift, impaired epidermal differentiation and disrupted barrier function in JunB conditional knockout mice. These findings define an unprecedented role for JunB in epidermal-pilosebaceous stem cell homeostasis and its pathology.

Activation of podocyte Notch mediates early Wt1 glomerulopathy

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger protein that regulates podocyte development and is highly expressed in mature podocytes. Mutations in the WT1 gene are associated with the development of renal failure due to the formation of scar tissue within glomeruli, the mechanisms of which are poorly understood. Here, we used a tamoxifen-based CRE-LoxP system to induce deletion of Wt1 in adult mice to investigate the mechanisms underlying evolution of glomerulosclerosis. Podocyte apoptosis was evident as early as the fourth day post-induction and increased during disease progression, supporting a role for Wt1 in mature podocyte survival. Podocyte Notch activation was evident at disease onset with upregulation of Notch1 and its transcriptional targets, including Nrarp. There was repression of podocyte FoxC2 and upregulation of Hey2 supporting a role for a Wt1/FoxC2/Notch transcriptional network in mature podocyte injury. The expression of cleaved Notch1 and HES1 proteins in podocytes of mutant mice was confirmed in early disease. Furthermore, induction of podocyte HES1 expression was associated with upregulation of genes implicated in epithelial mesenchymal transition, thereby suggesting that HES1 mediates podocyte EMT. Lastly, early pharmacological inhibition of Notch signaling ameliorated glomerular scarring and albuminuria. Thus, loss of Wt1 in mature podocytes modulates podocyte Notch activation, which could mediate early events in WT1-related glomerulosclerosis.

Oligodendrocyte RasG12V expressed in its endogenous locus disrupts myelin structure through increased MAPK, nitric oxide, and notch signaling.

Costello syndrome (CS) is a gain of function Rasopathy caused by heterozygous activating mutations in the HRAS gene. Patients show brain dysfunction that can include abnormal brain white matter. Transgenic activation of HRas in the entire mouse oligodendrocyte lineage resulted in myelin defects and behavioral abnormalities, suggesting roles for disrupted myelin in CS brain dysfunction. Here, we studied a mouse model in which the endogenous HRas gene is conditionally replaced by mutant HRasG12V in mature oligodendrocytes, to separate effects in mature myelinating cells from developmental events. Increased myelin thickness due to decompaction was detectable within one month of HRasG12V expression in the corpus callosum of adult mice. Increases in active ERK and Nitric Oxide (NO) were present in HRas mutants and inhibition of NO synthase (NOS) or MEK each partially rescued myelin decompaction. In addition, genetic or pharmacologic inhibition of Notch signaling improved myelin compaction. Complete rescue of myelin structure required dual drug treatments combining MAPK, NO, or Notch inhibition; with MEK + NOS blockade producing the most robust effect. We suggest that individual or concomitant blockade of these pathways in CS patients may improve aspects of brain function.

PlexinD1 Is a Novel Transcriptional Target and Effector of Notch Signaling in Cancer Cells

The secreted semaphorin Sema3E controls cell migration and invasiveness in cancer cells. Sema3E-receptor, PlexinD1, is frequently upregulated in melanoma, breast, colon, ovarian and prostate cancers; however, the mechanisms underlying PlexinD1 upregulation and the downstream events elicited in tumor cells are still unclear. Here we show that the canonical RBPjk-dependent Notch signaling cascade controls PlexinD1 expression in primary endothelial and cancer cells. Transcriptional activation was studied by quantitative PCR and promoter activity reporter assays. We found that Notch ligands and constitutively activated intracellular forms of Notch receptors upregulated PlexinD1 expression; conversely RNAi-based knock-down, or pharmacological inhibition of Notch signaling by gamma-secretase inhibitors, downregulated PlexinD1 levels. Notably, both Notch1 and Notch3 expression positively correlates with PlexinD1 levels in prostate cancer, as well as in other tumor types. In prostate cancer cells, Sema3E-PlexinD1 axis was previously reported to regulate migration; however, implicated mechanisms were not elucidated. Here we show that in these cells PlexinD1 activity induces the expression of the transcription factor Slug, downregulates E-cadherin levels and enhances cell migration. Moreover, our mechanistic data identify PlexinD1 as a pivotal mediator of this signaling axis downstream of Notch in prostate cancer cells. In fact, on one hand, PlexinD1 is required to mediate cell migration and E-cadherin regulation elicited by Notch. On the other hand, PlexinD1 upregulation is sufficient to induce prostate cancer cell migration and metastatic potential in mice, leading to functional rescue in the absence of Notch. In sum, our work identifies PlexinD1 as a novel transcriptional target induced by Notch signaling, and reveals its role promoting prostate cancer cell migration and downregulating E-cadherin levels in Slug-dependent manner. Collectively, these findings suggest that Notch-PlexinD1 signaling axis may be targeted to impair prostate cancer cell invasiveness and metastasis.