Gamma-secretase Research Articles

The in vivo Expression of Gamma secretase catalytic subunit (presenilin) in patients with chronic lymphocytic leukemia

Background:Gamma secretase is an intramembranous protease that plays an important role in NOTCHpathway that has a central role in malignancies Aim and objectives: The Aim of this work is to evaluate the in vivo expression of gamma secretase catalytic subunit (Presenilin) in patients with B cell lymphocytic leukemia. Subjects and Methods: This is a cross sectional study, was carried out at Internal medicine department, clinical hematology inpatient unit and outpatient department (OPD),Ain Shams University Hospitals. On 30 patients with chronic lymphocytic leukemia, between 2020 and 2021 Result:There was high statistically significant correlation between the gamma secretase catalytic subunit presenilin expression with type of patients being higher in relapsed and resistant cases than newly diagnosed.There was high statistically significant difference between the patient and healthy group as regard the gamma secretase gene catalytic subunit presenilinexpression Conclusion The gamma secretase catalytic subunit(presenilin) expression in cLL patients was elevated in comparision to healthy individuals and was elevated in resistant- relapsed cLL patients in comparision to newly diagnosed individuals and that will add prognostic role.So, gamma secretase inhibitors could be a light path as a line of therapy

Presenilin 1 is a direct regulator of the cardiac sarco/endoplasmic reticulum calcium pump

The gamma secretase catalytic subunit presenilin 1 (PS1) is expressed in the endoplasmic reticulum (ER) of neurons, where it regulates Ca2+ signaling. PS1 is also expressed in heart, but its role in regulation of cardiac Ca2+ transport remains unknown. Since the type 2 sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a) plays a central role in cardiac Ca2+ homeostasis, we studied whether PS1 regulates the cardiac SERCA2a function. The experiments were conducted in an inducible human SERCA2a stable T-Rex-293 cell line transfected with fluorescently labeled PS1 and the ER Ca2+ sensor R-CEPIA1er. Confocal imaging showed that that PS1 is localized predominantly in the ER membrane. Fluorescent resonance energy transfer (FRET) experiments in HEK293 cells transfected with fluorescently labeled SERCA2a and PS1 revealed that the two proteins directly interact with a 1:1 stoichiometry. The functional significance of this interaction was investigated in a heterologous cellular environment using a novel approach to directly measure ER Ca2+ dynamics. Measurements of SERCA2a-mediated Ca2+ transport showed that PS1 enhanced Ca2+ uptake at low ER Ca2+ loads (<0.15 mM) and reduced uptake at high loads (>0.35 mM). The results of this study revealed that PS1 could act as an important regulator of the cardiac Ca2+ pump function with a complex stimulatory/inhibitory profile.

1789P The gamma secretase inhibitor AL101 combined with other drugs for dual targeting of Notch dysregulated tumors

ACC & TNBC are tumors associated with Notch signaling pathway dysregulation. Ayala is targeting Notch activated tumors with investigational small molecule pan-Notch γ-secretase inhibitors AL101 & AL102. AL101 is currently being investigated in 2 phase II open-label, single-arm, multicenter studies in ACC (ACCURACY; NCT03691207) and TNBC (TENACITY; NCT04461600) in patients harboring known Notch1-4 activating alterations. Notch interacts via crosstalk with other signaling pathways, and here we describe testing for their identity to provide guidance for future combination studies with AL101.

904P ACCURACY: A phase II trial of AL101, a selective gamma secretase inhibitor, in subjects with recurrent/metastatic (R/M) adenoid cystic carcinoma (ACC) harboring Notch activating mutations (Notchmut): Results of 6-mg cohort

Notch signaling plays a key role in ACC tumorigenesis. Notchmut are found in ∼20% of ACC tumors, which are aggressive with a poor prognosis (Ferrarotto 2016, Ho 2019). No therapies are approved for R/M ACC. AL101, an investigational γ-secretase inhibitor, blocks Notch signaling and inhibits tumors in ACC patient-derived xenograft models with Notchmut (AACR ‘19, Abstr 4885). AL101 has clinical activity at 4 mg once weekly (QW) with a disease control rate of 68% (15% partial response) and appears to be well tolerated (Ferrarotto ESMO ‘20, #919MO).

Notch Signaling Pathway Is Activated by Sulfate Reducing Bacteria.

Sulfate Reducing Bacteria (SRB), usually rare residents of the gut, are often found in increased numbers (called a SRB bloom) in inflammatory conditions such as Inflammatory Bowel Disease (IBD), pouchitis, and periodontitis. However, the underlying mechanisms of this association remain largely unknown. Notch signaling, a conserved cell-cell communication pathway, is usually involved in tissue development and differentiation. Dysregulated Notch signaling is observed in inflammatory conditions such as IBD. Lipolysaccharide and pathogens also activate Notch pathway in macrophages. In this study, we tested whether Desulfovibrio, the most dominant SRB genus in the gut, may activate Notch signaling. RAW 264.7 macrophages were infected with Desulfovibrio vulgaris (DSV) and analyzed for the expression of Notch signaling pathway-related proteins. We found that DSV induced protein expression of Notch1 receptor, Notch intracellular domain (NICD) and p21, a downstream Notch target, in a dose-and time-dependent manner. DSV also induced the expression of pro-IL1β, a precursor of IL-1β, and SOCS3, a regulator of cytokine signaling. The gamma secretase inhibitor DAPT or Notch siRNA dampened DSV-induced Notch-related protein expression as well the expression of pro-IL1β and SOCS3. Induction of Notch-related proteins by DSV was not affected by TLR4 -IN -C34(C34), a TLR4 receptor antagonist. Additionally, cell-free supernatant of DSV-infected macrophages induced NICD expression in uninfected macrophages. DSV also activated Notch pathway in the human epithelial cell line HCT116 and in mouse small intestine. Thus, our study uncovers a novel mechanism by which SRB interact with host cells by activating Notch signaling pathway. Our study lays a framework for examining whether the Notch pathway induced by SRB contributes to inflammation in conditions associated with SRB bloom and whether it can be targeted as a therapeutic approach to treat these conditions.

Effects of the Gamma Secretase Inhibitors (GSI) Dapt (GSI-IX) and LSN424354 on Gene Expression and Shedding of Bcma from Multiple Myeloma and Chronic Lymphocytic Leukemia Tumor Cells

Introduction: Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) patients have high levels of solubilized (s) BCMA in the blood, and this protein is responsible for the immune deficiency of MM. sBCMA may also interfere with the efficacy of anti-BCMA-CAR-transduced T cells or other anti-BCMA antibody-based immunotherapy for multiple myeloma patients. Recently, γ-secretase has been identified as the enzyme that leads to the shedding of BCMA from B-cells. Gamma secretase (GS) is a multi-subunit protease complex that includes four individual proteins: presenilin-1 (PSEN1), nicastrin, anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). CD147 acts as a non-essential regulator of the complex. We examined gene expression of PSEN1, APH-1, PEN-2, and CD147 among MM pts with progressive disease (PD) and complete remission (CR). We also determined the effects of the GS inhibitors (i) DAPT (GSI-IX) and LSN424354 (Eli Lilly, Indianapolis, IN) on solubilized BCMA levels from cultured human MM and CLL tumor cells.Study design: Bone marrow (BM) mononuclear cells (MCs) and serum were collected from monoclonal gammopathy of undetermined significance (MGUS) individuals, MM patients and healthy subjects after obtaining IRB approval. BMMCs from MM patients were treated with or without DAPT (GSI-IX) in a concentration dependent fashion for 24 hours. Following incubation, cells were washed three times using PBS. Flow cytometric analysis was performed using a Beckman Coulter Cytomics FC 500 with Cytomics CXP software. Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Louisville, KY) following the manufacturer's instructions. Quantitative PCR (qPCR) was applied to measure the relative abundance of human PSEN1, APH-1, PEN-2, and CD147 mRNA compared to that of the housekeeping gene HPRT mRNA. Single-cell suspensions were prepared from human MM LAGκ-1A xenografts which had been grown in the left superficial gluteal muscle of SCID mice. Cells were cultured and treated with the LSN424354 in a concentration dependent fashion. Supernatant BCMA levels were determined using an ELISA (R&D Systems, Minneapolis, MN) assay.Results and Discussion: qPCR showed PEN-2 gene expression was only slightly increased among MM patients with progressive disease (PD) compared to those in complete response or normal subjects whereas there was no change in expression of the PSEN1 or APH1 genes. CD147 gene expression was markedly increased in MM pts in PD (n=25) compared with those in CR (n=18; P=0.005) or MGUS (n=9; P=0.005). Next, we examined the effect of the DAPT (GSI-IX) on primary human MM tumor cells. Supernatants from primary human MM cells cultured for 24 hours in DAPT showed markedly reduced BCMA in a concentration dependent fashion (range,0.2 nM-2µM). Using qPCR, DAPT markedly reduced PEN-2 gene expression in a concentration dependent fashion. On the other hand, this GSI did not effect BCMA gene expression. DAPT reduces shedding of BCMA from B-cells but not its gene expression. We also determined BCMA levels in supernatants from cultured tumor cells derived from the human MM LAGκ-1A xenograft following 5 days of tissue culture with LSN424354 at concentrations ranging from 100 pM to 10 nM. Supernatant sBCMA levels were reduced > 90% at the highest concentrations (10nM). Similarly, freshly obtained CLL tumor cells cultured with LSN424354 at concentrations ranging from 100 pM to 10 nM also showed a marked reduction in supernatant BCMA levels at the higher concentrations.Conclusion: Gamma secretase sheds BCMA off B-cells. CD147, a regulator of gamma secretase activity, shows markedly higher gene expression among MM pts with PD compared with those in CR or MGUS individuals. The GSIs DAPT and LSN424354 reduce shedding of BCMA from malignant cells from MM and CLL patients. Since GSIs reduce solubilized BCMA levels, it is possible that treatment with these agents can reverse the immune dysfunction caused by this solubilized protein, and also improve the efficacy of anti-BCMA immunotherapies for treating patients with multiple myeloma and CLL. DisclosuresBerenson:Incyte: Consultancy, Research Funding; OncoTracker, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Notch Signaling Controls Drug Response in B-Cell Acute Lymphoblastic Leukemia (B-ALL)

▪Background: Growing evidences suggest that Notch signaling can modulate drug response in hematological malignancies including T-ALL, B-CLL and AML. In B-cell acute lymphoblastic leukemia (B-ALL) we have previously demonstrated that Notch3 and Notch4 support survival of primary B-ALL cells, precluding a role of Notch signaling in drug response.Aims: In this study we used in vitro, in silico and in vivo experiments to comprehensively define the role of Notch signaling in B-ALL pathogenesis in terms of prognosis, proliferation survival and drug response.Methods: B-ALL cell lines were obtained from ATCC, while B-ALL primary cells were obtained from bone marrow or peripheral blood of 45 B-ALL patients. Flow cytometry and western immunoblotting were used to study the expression of Notch receptors and ligands. Drugs used were Cytarabine (Ara-C), Dexamethasone (Dexa) and Doxorubicin (Doxo) alone or in combination with Notch modulators including anti-Notch blocking antibodies, gamma secretase inhibitors (GSIs), and Notch transcription factor inhibitor (SAHM1). Mice xenograft model of B-ALL were obtained by injecting the B-ALL line RS4;11 in NOD/Shi-scid/IL-2Rγnull mice (NOG). Cell viability was evaluated by Annexin-V/PI and MTT assay; proliferation was assessed through CFSE dilution.Results: Our data revealed a robust expression of Notch receptors and ligands in B-ALL primary cells and B-ALL cell lines. The expression pattern consisted in high expression levels of Notch1, Notch3, Notch4, Jagged2, DLL3 and DLL4. Notably, in primary blast cells deriving from patients, the expression of Notch3, Notch4 and Jagged2, were significantly higher in the cases refractory to treatment as compared to patients achieving complete remission. Having also observed in samples derived from patients after chemotherapy a reduction in expression levels of Notch1-4 compared to samples collected prior the treatment, we hypothesized that Notch signaling was critical to drug response in B-ALL. Analysis of B-ALL cells treated with conventional chemo-agents (Ara-C, Doxo and Dexa) alone or in combination with Notch signaling inhibitors, showed that Notch signaling inhibitors including GSIs and anti-Notch4 were all able to significantly potentiate drug-induced cell death in B-ALL cells, up-regulating intracellular levels of reactive oxygen species (ROS). These high intracellular levels or ROS were then capable to modulate prosurvival-protein levels such as mTor, Akt, NFκ-B and Erk. In vitro observations were successfully translated in NOG mice, where GSI-XII in association with Ara-C significantly lowered leukemic burden in bone marrow of xenograft models of B-ALL, prolonging survival of mice compared to DMSO or Ara-C alone.Conclusion: In this study, we highlighted the prognostic value of Notch expression in B-ALL as well as its critical role in B-ALL cell survival and response to chemotherapy in vitro and in vivo ; we demonstrated that Notch inhibitors can improve Ara-C-mediated reduction of blast cells in bone marrow, revealing that Notch signaling is a possible therapeutic strategy to eradicate minimal residual disease in B-ALL. Overall, our results support further investigations of Notch inhibitors in clinical trials of B-ALL. DisclosuresBonifacio:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Notch Signaling Promotes the Differentiation of Human CLEC9A+ Dendritic Cells from Hematopoietic Stem and Progenitor Cells

Human CLEC9A+ dendritic cells (also known as CD141+ DCs, XCR1+ DCs, and cDC1) are an endogenous DC subset with constitutive ability to traffic to secondary lymphoid tissues, efficiently cross-present cellular antigens, and prime CD4+ and CD8+ antiviral and antitumor T cell responses. As such, CLEC9A+ DCs have been proposed as ideal candidates for adoptive cell immunotherapy, however their rarity in vivo and a lack of knowledge regarding their development have thus far precluded their therapeutic use.We used in vitro differentiation approaches to identify novel microenvironmental cues that regulate the development of CLEC9A+ DCs from human CD34+ hematopoietic stem and progenitor cells (HSPCs). Our findings reveal a previously unknown role for Notch signaling as a key regulatory pathway in CLEC9A+ DC differentiation from HSPCs. Exposure of HSPCs to the Notch ligand DLL1 expressed by MS5 stromal cells, together with the DC-promoting cytokines FLT3L and GM-CSF, markedly enhanced the generation of CLEC9A+ DCs relative to control cultures lacking DLL1 expression. Resulting CLEC9A+ DC frequency was on average 66% of total cells in DLL1 cultures, compared with 6% in control cultures at 20 days (p < 0.0001). Promotion of CLEC9A+ DC development was accompanied by statistically significant decreases in monocyte, CD1c+ DC, and plasmacytoid DC frequencies and cell numbers, with granulocytic differentiation relatively unaffected. Similar changes were seen when DLL1 was substituted for the Notch ligands DLL4 or JAG1, and abrogated by gamma secretase inhibition. Furthermore, promotion of CLEC9A+ DC differentiation was mimicked by transduction of HSPCs with intracellular Notch1, consistent with a cell-intrinsic role for Notch in CLEC9A+ DC development.The effect of Notch signaling on CLEC9A+ DC development was conserved between HSPCs isolated from human cord blood (CB), bone marrow (BM), or G-CSF mobilized peripheral blood (MPB). Furthermore, highly purified BM monocyte/dendritic cell progenitors (MDP) or common dendritic cell progenitors (CDP) cultured in the presence of DLL1 preferentially generated CLEC9A+ DCs with loss of alternative lineage outputs, supporting a mechanism by which Notch signaling skews lineage commitment to the CLEC9A+ DC lineage at the level of multipotent DC progenitors.Notch-induced CLEC9A+ DCs derived from HSPCs were phenotypically and functionally similar to primary CLEC9A+ DCs isolated from the blood. Whole transcriptome profiling by RNAseq of Notch-induced CLEC9A+ DCs confirmed lineage-specific upregulation of key CLEC9A+ DC genes including XCR1, CADM1, BATF3, and IRF8 . Furthermore, global gene expression profiles were similar between Notch-induced CLEC9A+ DCs generated from HSPCs isolated from CB or MPB. Notch-induced CLEC9A+ DCs expressed high levels of CD62L and CCR7, and underwent efficient chemotaxis in response to CCL21 in transwell migration assays, suggesting lymph node-homing capacity. Compared to primary blood CLEC9A+ DCs, Notch-induced CLEC9A+ DCs exhibited higher basal surface expression of T cell costimulatory molecules including CD80, CD83, and CD86, reminiscent of dermal CLEC9A+ DCs, and these were further upregulated in response to the TLR agonists poly(I:C) or R848. Notch-induced CLEC9A+ DCs exhibited potent CD4+ and CD8+ immunostimulatory activity in mixed lymphocyte reactions, and induced antigen-specific CD8+ T cell responses to an HLA-A*0201-restricted NY-ESO-1 epitope through either cross-presentation of cellular antigen acquired from necrotic tumor cells, or endogenous presentation of antigen transduced at the CD34+ HSPC stage.In conclusion, we have identified a novel role for Notch signaling in the differentiation of CLEC9A+ DCs from HSPCs. We propose Notch signaling enforces CLEC9A+ DC lineage commitment in multipotent DC progenitors, and RNAseq studies addressing the molecular basis of Notch-induced CLEC9A+ DC commitment are in progress. Importantly, the identification of Notch as a critical regulatory pathway in human CLEC9A+ DC development allows robust in vitro differentiation of large numbers of highly functional CLEC9A+ DCs. Taken together our findings provide insight into the development of this important DC lineage and permit the preclinical development of next generation cellular vaccine strategies using in vitro derived CLEC9A+ DCs. DisclosuresMontel-Hagen:Kite Pharma: Research Funding. Kohn:Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biogen IDEC: Research Funding; BioMarin Pharmaceutical: Research Funding; Orchard Therapeutics Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Crooks:Kite Pharma: Research Funding.

CME Dermatology 23/Answers: Hidradenitis suppurativa

CME Dermatology 23/Answers: Hidradenitis suppurativa Abstract. Hidradenitis suppurativa (Acne inversa) is a chronic skin disease characterized by recurrent inflammatory nodules, abscesses, and scarring mainly in the intertriginous areas. The prevalence is estimated to be 0.05 to 4.1 % worldwide, with women being more commonly affected. The exact pathogenesis remains unknown, but hair follicle occlusion and inflammation seem to be the initiating events. Several epidemiological correlations with smoking and obesity as well as mutations in gamma secretase genes were described. The therapy depends on the extension of the disorder and should respect the current guidelines. Topical clindamycin is suitable for mild forms of the disease whereas moderate to severe disease requires systemic anti-inflammatory treatment. Surgical intervention is currently the only definitive treatment for hidradenitis suppurativa.

A patient reported outcomes of treatments for desmoid tumors: An international natural history study.

11560 Background: The Desmoid Tumor Research Foundation (DTRF) launched the natural history study (NHS) in 2017. At this time, there are no standard-of-care options for this rare sarcoma. The treatments, clinical descriptors, and the patient reported outcomes to pharmacologic agents are described here within. Methods: The web-based natural history study launched September 2017 in collaboration with the National Organization of Rare Disorders. It contains 15 surveys covering diagnostics, disease, treatment, care management, and quality of life. Treatment types included in the DTRF NHS were pharmacology, surgery, radiation, high-intensity focused ultrasound (HiFU), and active surveillance (watch and wait). Results: While surgery was once the primary intervention for desmoid tumor patients, the NHS participants reported that 47.6% had received active surveillance or no systemic treatment at diagnosis. This is most common for desmoid tumors located in abdominal wall (54/103; 52.4%). There were 87 reported cases of complete surgical resection, 38 incomplete resections, and 23 bowel resections. 9 amputations were reported; 8 participants reported recurrent disease following the removal of the limb. The non-surgical interventions, such as radiation and HiFU, were mostly described for participants with chest wall tumors (15 pts) and joints/extremities (10 pts). Many options for systemic therapies were described including sorafenib (44/284; 15.5%), sulindac (36/284; 12.7%), and anti-hormonal agents tamoxifen and toremifene (34/285; 10.9%) were described. Targeted agents, such as gamma secretase inhibitor, pazopanib, and sorafenib, were greater in the United States than the non-US country participants (21% vs 9%). Multiple lines of treatments were reported by 81 participants, surgery is greatest as the first intervention for all tumor locations (49/81, 60%), with the exception of those with head/neck tumors who received chemotherapy (6/11, 55%). Analysis has started to evaluate the efficacy of systemic treatments from these NHS data. The table describes the participant reported outcomes of anti-hormonal agents, chemotherapeutics, non-steroidal anti-inflammatories, and targeted agents. Both chemotherapies and targeted agents were reported to have 38.1% response rates from the participants with 34.3% and 23.8% of participants reported progressive disease on therapy, respectively. Conclusions: Desmoid tumor NHS study participants reported the use of many treatment modalities demonstrating a range of frequency of use by tumor location and efficacy. Data collection through the DTRF NHS is ongoing.[Table: see text]

CME Dermatology 23: Hidradenitis suppurativa

CME Dermatology 23: Hidradenitis suppurativa Abstract. Hidradenitis suppurativa (Acne inversa) is a chronic skin disease characterized by recurrent inflammatory nodules, abscesses, and scarring mainly in the intertriginous areas. The prevalence is estimated to be 0.05 to 4.1 % worldwide, with women being more commonly affected. The exact pathogenesis remains unknown, but hair follicle occlusion and inflammation seem to be the initiating events. Several epidemiological correlations with smoking and obesity as well as mutations in gamma secretase genes were described. The therapy depends on the extension of the disorder and should respect the current guidelines. Topical clindamycin is suitable for mild forms of the disease whereas moderate to severe disease requires systemic anti-inflammatory treatment. Surgical intervention is currently the only definitive treatment for hidradenitis suppurativa.

Investigation of gamma secretase gene complex mutations in German population with Hidradenitis suppurativa designate a complex polygenic heritage.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease affecting apocrine gland-bearing skin in the axilla, groin and under the breasts. Mutations of the gamma secretase gene complex, which is essential in the activation of Notch signalling pathways, were shown in some families with HS and in a few sporadic cases. Although an imbalance in Notch signalling is implicated in the pathogenesis, the exact mechanism of HS development is yet unknown. We aim to investigate the genetic basis of HS by determining the presence of mutations of gamma secretase gene complex in a cohort of HS patients and by searching for a disease-causing pathogenic variant in a multi-generational HS family using parametric linkage analysis. Thirty-eight patients clinically diagnosed with HS were included in this study. All exons and exon-intron boundaries of the genes encoding gamma secretase complex consisting of six genes: APH1A, APH1B, PSENEN, NCSTN, PSEN1 and PSEN2 were sequenced by Sanger technique. Genetic mapping with parametric linkage analysis for the patients in the family was performed with eight affected and four healthy individuals. The logarithm of odds was calculated. In a sporadic patient with early-onset, severe lesions in axilla and groin, a novel single-nucleotide deletion causing frameshift in exon 1 of the NCSTN gene was identified ((NM_015331.3): c.38delG, p.(Gly13Glufs*15)). The LOD score of 1.5 was never exceeded in any region of the genome, pointing towards intricate multi-genic inheritance pattern within the affected family. The gamma secretase gene complex mutations were rare in our cohort (3.2%). Besides, our analysis indicates a possible complex multi-genic inheritance in a seemingly autosomal dominantly inherited large HS family. Genetics of both familial and sporadic HS may be complicated in most cases, and the role of other potential genes such as autoinflammatory and modifier genes as well as environmental factors may influence the pathogenesis.

Gamma Secretase Inhibitor: Therapeutic Target via NOTCH Signaling in T Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a diseased condition of bone marrow and lymphoblast, mainly expressed on T-cell immune phenotype. Diagnosis of TALL patients shows the burden of a large tumour and leukemia cells in the peripheral blood vessel which often infiltrates into the central nervous system. Chemotherapy is considered the primary mode of treatment for this disease, but recent advancements in the molecular understanding of the disease, including NOTCH1 signaling, could offer some alternatives. NOTCH signaling undergoes a non-regulated mutation at NOTCH1 in most T-ALL. Gamma-secretase (GS) plays a key role in blocking of proteolytic activation of NOTCH receptors, which could potentially be a new targeted therapy for this type of leukaemia. This study mainly aims to outline the role of γ-secretase inhibitors via NOTCH signaling in TALL. The role of GSI (γ-secretase inhibitor) in most T-ALL cell lines has been linked to the targeting pathway of NOTCH signaling. Mutation at NOTCH1 has however not served as a predictor of γ-secretase inhibitor sensitivity because of several factors, including gene expression of NOTCH pathway activity. Therefore, despite the promising outcome of this approach in NOTCH-1 activated T-ALL, not all patients with this condition are expected to respond. Long-term therapeutic success against cancerous cells is rarely achieved with monotherapy, and even targeting investigational pathways such as NOTCH may require a combination regimen. Ultimately, the optimised use of these new therapeutic agents may become the next tool in our search for an effective 'individualized medicine'.

Recruitment and retention of participant and study partner dyads in two multinational Alzheimer\u2019s disease registration trials

BackgroundEarly study exit is detrimental to statistical power and increases the risk for bias in Alzheimer’s disease clinical trials. Previous analyses in early phase academic trials demonstrated associations between rates of trial incompletion and participants’ study partner type, with participants enrolling with non-spouse study partners being at greater risk.MethodsWe conducted secondary analyses of two multinational phase III trials of semagacestat, an oral gamma secretase inhibitor, for mild-to-moderate AD dementia.Cox’s proportional hazards regression model was used to estimate the relationship between study partner type and the risk of early exit from the trial after adjustment for a priori identified potential confounding factors. Additionally, we used a random forest model to identify top predictors of dropout.ResultsAmong participants with spousal, adult child, and other study partners, respectively, 35%, 38%, and 36% dropped out or died prior to protocol-defined study completion, respectively. In unadjusted models, the risk of trial incompletion differed by study partner type (unadjusted p value = 0.027 for test of differences by partner type), but in models adjusting for potential confounding factors, the differences were not statistically significant (p value = 0.928). In exploratory modeling, participant age was identified as the primary characteristic to explain the relationship between study partner type and the risk of failing to complete the trial. Participant age was also the strongest predictor of trial incompletion in the random forest model.ConclusionsAfter adjustment for age, no differences in the risk of incompletion were observed when comparing participants with different study partner types in these trials. Differences between our findings and the findings of previous studies may be explained by differences in trial phase, size, geographic regions, or the composition of academic and non-academic sites.

Gamma Secretase Inhibitors in Cancer: A Current Perspective on Clinical Performance.

Gamma secretase inhibitors (GSIs), initially developed as Alzheimer's therapies, have been repurposed as anticancer agents given their inhibition of Notch receptor cleavage. The success of GSIs in preclinical models has been ascribed to induction of cancer stem-like cell differentiation and apoptosis, while also impairing epithelial-to-mesenchymal transition and sensitizing cells to traditional chemoradiotherapies. The promise of these agents has yet to be realized in the clinic, however, as GSIs have failed to demonstrate clinical benefit in most solid tumors with the notable exceptions of CNS malignancies and desmoid tumors. Disappointing clinical performance to date reflects important questions that remain to be answered. For example, what is the net impact of these agents on antitumor immune responses, and will they require concurrent targeting of tumor-intrinsic compensatory pathways? Addressing these limitations in our current understanding of GSI mechanisms will undoubtedly facilitate their rational incorporation into combinatorial strategies and provide a valuable tool with which to combat Notch-dependent cancers. In the present review, we provide a current understanding of GSI mechanisms, discuss clinical performance to date, and suggest areas for future investigation that might maximize the utility of these agents. IMPLICATIONS FOR PRACTICE: The performance of gamma secretase inhibitors (GSIs) in clinical trials generally has not reflected their encouraging performance in preclinical studies. This review provides a current perspective on the clinical performance of GSIs across various solid tumor types alongside putative mechanisms of antitumor activity. Through exploration of outstanding gaps in knowledge as well as reasons for success in certain cancer types, the authors identify areas for future investigation that will likely enable incorporation of GSIs into rational combinatorial strategies for superior tumor control and patient outcomes.

Notch signaling regulates function of human mucosal-associated invariant T (MAIT) cells.

Notch signaling plays an important role in the development of T lymphocytes and regulates their effector functions. The regulatory roles of Notch signaling on T cells have been intensely investigated, but whether it involves in effector functions of mucosal-associated invariant T (MAIT) cells has never been reported. To elucidate the expression profiles of Notch receptors/ligands and to investigate their roles in human MAIT cell function. Peripheral blood mononuclear cells (PBMCs) from health donors were stimulated with or without anti-CD3/ CD28-coupled beads, recombinant IL-12/IL-18 cytokines, riboflavin- or non-riboflavin-synthesizing bacterial cultured supernatant for 24 hours. The expression profiles of Notch receptors and ligands on MAIT cells were detected by flow cytometry. PBMCs were treated with a Notch signaling inhibitor, gamma secretase inhibitor (GSI), before stimulation to investigate the impact of interfering with Notch signaling on activation and function of MAIT cells. Resting MAIT cells predominantly expressed Notch2 receptor and the ligand, Jagged 2, on their surface. Upon stimulation, MAIT cells further upregulated Notch2 and also Notch1 with its cleaved form, indicating active Notch signaling. Cytokines and cytotoxic molecules which are secreted by activated MAIT cells, were suppressed by treatment with GSI. Moreover, both TCR-dependent MAIT cell activation by microbial-derived riboflavin intermediates and TCR-independent MAIT cell activation driven by IL-18 in synergy with IL-12, were blocked by GSI treatment. Notch signaling is operating in MAIT cells and is involved in their activation both in a TCR-independent and -dependent manners.

Insight γ-Secretase: Structure, Function, and Role in Alzheimer's Disease.

In neurodegenerative disorders, there is a progressive degeneration of the body, leading to the death of nerve cells. In this state, a patient gets affected day by day with mental weakness, dementia, and ataxia. Alzheimer's disease (AD) is the most common irreversible neurodegenerative brain disorder mainly affecting people over the age of 65. Many types of research suggest that the main culprit for AD is the aggregated form of a (39-43) amino acid peptide called amyloid beta. Amyloid beta (Aβ) is generated by the action of beta-secretase and gamma-secretase on the larger glycoprotein. Gamma (γ) secretase is an intra-membrane protease complex that cleaves the single-- pass transmembrane protein, the amyloid precursor protein, and Notch. The γ-secretase complex contains presenilin, presenilin enhancer-2, anterior pharynx defective-1, and nicastrin. Any mutation in presenilin-1 or the cleavage of amyloid precursor protein by γ-secretase directly or indirectly is associated with AD. Therefore, the prevention of this enzyme is one of the solutions for AD. In this article, we discuss the γ-secretase complex and its inhibitors that can contribute to the prevention of AD.

NOTCH signalling in ovarian cancer angiogenesis.

The Notch signalling pathway is involved in the new vessel formation process by regulating tip and stalk cells, which are key cells in the sprout formation. This process is essential in both normal ovary and cancer angiogenesis and is regulated by Notch-VEGF crosstalk. Furthermore, Notch has been linked in ovary with stem cell maintenance and epithelial mesenchymal transition processes. Dysregulation of the Notch pathway is frequent in ovarian cancer (OC) and it has been associated with impaired survival and advanced stages or lymph node involvement. Notch also plays a role in chemoresistance to platinum. In this context, this pathway has emerged as an attractive target for precision medicine in OC. Two main targets of this pathway concentrate the clinical development of compounds blocking Notch: gamma secretase and Delta-like ligand 4. Most of the clinical trials including OC patients have been developed in phase I or phase Ib. Despite being in an early phase, both of these compounds, navicixizumab or demcizumab, two monoclonal antibodies targeting Dll4, showed promising efficacy data in platinum-resistant OC patients in recent studies. This review will focus on the mechanisms of the Notch pathway with special interest in angiogenesis regulation and the implication of Notch as a potential therapeutic target in OC.

Long non-coding RNA HOTAIR induces GLI2 expression through Notch signalling in systemic sclerosis dermal fibroblasts

ObjectivesSystemic sclerosis (SSc) is characterised by tissue fibrosis of the major organs of the body including the skin, lungs and heart. We have previously reported that the lncRNA HOTAIR plays a central role in the activation of SSc myofibroblasts, the key cellular elements of fibrosis. HOTAIR induces fibroblast activation through H3K27me3-mediated activation of the Notch signalling pathway. Here we aimed to identify the signalling events downstream of Notch that drive SSc myofibroblast activation.MethodsPatient fibroblasts were obtained from full-thickness forearm skin biopsies of 3 adult patients with SSc of recent onset. The lncRNA HOTAIR was expressed in healthy dermal fibroblasts by lentiviral transduction. Hedgehog signalling pathway was inhibited with GANT61 and GLI2 siRNA. Gamma secretase inhibitors RO4929097 and DAPT were used to block Notch signalling. GSK126 was used to inhibit Enhancer of Zeste 2 (EZH2).ResultsOverexpression of HOTAIR in dermal fibroblasts induced the expression of the Hedgehog pathway transcription factor GLI2. This is mediated by activation of Notch signalling following epigenetic downregulation of miRNA-34a expression. Inhibition of H3K27 methylation and Notch signalling reduced expression of GLI2 in HOTAIR-expressing fibroblasts as well as in SSc dermal fibroblasts. Importantly, the inhibition of GLI2 function using GANT61 or siRNA mitigates the pro-fibrotic phenotype induced by HOTAIR.ConclusionsOur data indicates that GLI2 expression is stably upregulated in SSc myofibroblasts through HOTAIR and that GLI2 mediates the expression of pro-fibrotic markers downstream of Notch.

In Vitro Evaluation of Notch Inhibition to Enhance Efficacy of Radiation Therapy in Melanoma

PurposeThe scope of radiation therapy is limited in melanoma. Using in vitro melanoma models, we investigated a Notch signaling inhibitor as a radiosensitizer to explore its potential to improve the efficacy of radiation therapy to widen the clinical application of radiation therapy in melanoma. Methods and MaterialsMelanoma cell lines A375, SKMEL28, and G361 were grown using standard tissue culture methods. Radiation was delivered with a clinical x-ray unit, and a gamma secretase inhibitor RO4929097 was used to inhibit Notch signaling. Cell viability signal was used to calculate Loewe’s combination index to assess the interaction between radiation and RO4929097 and also the effect of scheduling of radiation and RO4929097 on synergy. Clonogenic assays were used to assess the clonogenic potential. An in vitro 3-dimensional culture model, γ-H2AX, and notch intracellular domain assays were used to interrogate potential underlying biological mechanisms of this approach. Scratch and transwell migration assays were used to assess cell migration. ResultsA375 and SKMEL28 cell lines showed consistent synergy for most single radiation doses examined, with a tendency for better synergy with the radiation-first schedule (irradiation performed 24 hours before RO4929097 exposure). Clonogenic assays showed dose-dependent reduction in colony numbers. Both radiation and RO4929097 reduced the size of melanospheres grown in 3-dimensional culture in vitro, where RO4929097 demonstrated a significant effect on the size of A375 and SKMEL28 melanospheres, indicating potential modulation of stem cell phenotype. Radiation induced γ-H2AX foci signal levels were reduced after exposure to RO4929097 with a tendency toward reduction in notch intracellular domain levels for all 3 cell lines. RO4929097 impaired both de novo and radiation-enhanced cell migration. ConclusionsWe demonstrate Notch signaling inhibition with RO4929097 as a promising strategy to potentially improve the efficacy of radiation therapy in melanoma. This strategy warrants further validation in vivo.