Notch Inhibition Research Articles

Notch signaling in breast cancer: From pathway analysis to therapy.

The Notch signaling pathway, which is highly conserved from sea urchins to humans, plays an important role in cell-differentiation, survival, proliferation, stem-cell renewal, and determining cell fate during development and morphogenesis. It is well established that signaling pathways are dysregulated in a wide-range of diseases, including human malignancies. Studies suggest that the dysregulation of the Notch pathway contributes to carcinogenesis, cancer stem cell renewal, angiogenesis, and chemo-resistance. Elevated levels of Notch receptors and ligands have been associated with cancer-progression and poor survival. Furthermore, the Notch signaling pathway regulates the transcriptional activity of key target genes through crosstalk with several other signaling pathways. Indeed, increasing evidence suggests that the Notch signaling pathway may serve as a therapeutic target for the treatment of several cancers, including breast cancer. Researchers have demonstrated the anti-tumor properties of Notch inhibitors in various cancer types. Currently, Notch inhibitors are being evaluated for anticancer efficacy in a number of clinical-trials. However, because there are multiple Notch receptors that can exhibit either oncogenic or tumor-suppressing roles in various cells, it is important that the Notch inhibitors are specific to particular receptors that are tumorigenic in nature. This review critically evaluates existing Notch inhibitory drugs and strategies and summarizes the previous discoveries, current understandings, and recent developments in support of Notch receptors as therapeutic targets in breast cancer.

Small molecules re-establish neural cell fate of human fibroblasts via autophagy activation.

The generation of neural cells is of great interest in medical research because of its promising in neurodegenerative diseases. Small chemical molecules have been used for inducing specific cell types across lineage boundaries. Therefore, to direct neural cell fate, small molecule is a feasible approach for generating clinically relevant cell types without genetic alterations. Human fibroblasts have been directly induced into neural cells with different combinations of small molecules; however, the mechanism underlying neural induction is still not fully understood. In this study, human fibroblasts were induced into neural cells by using only 4 small molecules in a short time period, 5d. Small molecules used in this study included WNT activator, DNMT inhibitor, Notch inhibitor, and retinoic acid. Neural-specific genes, including NESTIN, TUJ1, and SOX2, were upregulated upon the induction for 5d. Noteworthy, this neural induction process by small molecules coincided with the activation of autophagy. Autophagy-related genes, such as LC3, ATG12, and LAMP1, were enhanced upon neural induction, and the number of induced-neural cells decreased when autophagy was suppressed by chloroquine. The activation of autophagy was found to reduce ROS generation within the induced-neural cells, and the inhibition of autophagy by chloroquine suppressed the expression of antioxidant genes, CATALASE, SOD, and GPX. This implied that autophagy maintained the optimal level of ROS for neural induction of human fibroblasts. Altogether, this study presented the effective and convenient condition to induce neural cells from human fibroblasts and revealed the positive roles of autophagy in controlling neural cell induction.

Notch Inhibition Prevents Differentiation of Human Limbal Stem/Progenitor Cells in vitro

Notch signaling has been shown to regulate the homeostasis and wound healing of the corneal epithelium. We investigated the effect of Notch inhibition in the human limbal stem/progenitor cells (LSCs) in vitro by using small molecules. Treatment of the LSCs with DAPT and SAHM1 reduced the proliferation rate and maintained the undifferentiated state of the LSCs in a concentration dependent manner. Stratification and differentiation of the corneal epithelium were not reduced after Notch inhibition, indicating that the function of the corneal basal cells is retained. Our findings suggest that Notch signaling plays a role in the proliferation and maintenance of LSCs.

Notch pathway in ependymoma RELA-fused subgroup: upregulation and association with cancer stem cells markers expression.

RELA-fused supratentorial (ST) ependymoma (EPN) is an aggressive subgroup with poor prognosis. Considering the putative role of Notch signaling in the maintenance of the cancer stem cells (CSC) phenotype in RELA-fused EPN, we investigated the expression of Notch pathway and its target genes in this subgroup. We also evaluated the effects of two Notch inhibitors (DAPT and RO4929097) on cell proliferation, apoptosis, colony formation, and CSCs markers gene expression on EPN cell line of the RELA-fused subgroup (BXD-1425). In addition, in silico signatures of the Notch genes and CSCs markers were analyzed on a large clinical dataset from GSE64415 study. We found that among the ST-EPN subgroups the Notch signaling (NOTCH1, JAG1, JAG2, and HES4) is specifically activated in the ST-EPN-RELA. Furthermore, treatment of the RELA-fused EPN cell line with the Notch inhibitors impaired the Notch signaling expression and revealed that Notch axis is not essential for cell proliferation and survival in this setting. NOTCH1 expression in ST-EPN was correlated with the CSCs markers VEGFA and L1CAM overexpression and JAG1 expression was correlated with the CCND1 and CDK6 overexpression. In addition, in vitro treatment with Notch inhibitors induced downregulation of CSCs markers. These findings indicate that Notch signaling can be involved in the ST-EPN-RELA CSCs maintenance by modulating the expression of genes responsible for cell phenotype and cell fate.

Early Notch Signals Induce a Pathogenic Molecular Signature during Priming of Alloantigen-Specific Conventional CD4+ T Cells in Graft-versus-Host Disease.

Graft-versus-host disease (GVHD) is the most serious complication of allogeneic hematopoietic cell transplantation. Notch signals delivered during the first 48 h after transplantation drive proinflammatory cytokine production in conventional T cells (Tconv) and inhibit the expansion of regulatory T cells (Tregs). Short-term Notch inhibition induces long-term GVHD protection. However, it remains unknown whether Notch blockade blunts GVHD through its effects on Tconv, Tregs, or both and what early Notch-regulated molecular events occur in alloantigen-specific T cells. To address these questions, we engineered T cell grafts to achieve selective Notch blockade in Tconv versus Tregs and evaluated their capacity to trigger GVHD in mice. Notch blockade in Tconv was essential for GVHD protection as GVHD severity was similar in the recipients of wild-type Tconv combined with Notch-deprived versus wild-type Tregs. To identify the impact of Notch signaling on the earliest steps of T cell activation in vivo, we established a new acute GVHD model mediated by clonal alloantigen-specific 4C CD4+ Tconv. Notch-deprived 4C T cells had preserved early steps of activation, IL-2 production, proliferation, and Th cell polarization. In contrast, Notch inhibition dampened IFN-γ and IL-17 production, diminished mTORC1 and ERK1/2 activation, and impaired transcription of a subset of Myc-regulated genes. The distinct Notch-regulated signature had minimal overlap with known Notch targets in T cell leukemia and developing T cells, highlighting the specific impact of Notch signaling in mature T cells. Our findings uncover a unique molecular program associated with the pathogenic effects of Notch in T cells at the earliest stages of GVHD.

Estrogen Signaling Drives Ciliogenesis in Human Endometrial Organoids.

The human endometrium is the inner lining of the uterus consisting of stromal and epithelial (secretory and ciliated) cells. It undergoes a hormonally regulated monthly cycle of growth, differentiation, and desquamation. However, how these cyclic changes control the balance between secretory and ciliated cells remains unclear. Here, we established endometrial organoids to investigate the estrogen (E2)-driven control of cell fate decisions in human endometrial epithelium. We demonstrate that they preserve the structure, expression patterns, secretory properties, and E2 responsiveness of their tissue of origin. Next, we show that the induction of ciliated cells is orchestrated by the coordinated action of E2 and NOTCH signaling. Although E2 is the primary driver, inhibition of NOTCH signaling provides a permissive environment. However, inhibition of NOTCH alone is not sufficient to trigger ciliogenesis. Overall, we provide insights into endometrial biology and propose endometrial organoids as a robust and powerful model for studying ciliogenesis in vitro.

Notch-1 inhibition reduces proliferation and promotes osteogenic differentiation of bone marrow mesenchymal stem cells.

Low differentiation and high proliferation rates are critical factors affecting bone marrow mesenchymal stem cell (BMSC) tumorigenesis. The present study aimed to investigate the role of the Notch signaling pathway in BMSC proliferation and osteogenic differentiation. Mouse BMSCs were divided into control, vector, Notch1-small interfering (si)RNA, γ-secretase inhibitor, and Notch1-siRNA + γ-secretase inhibitor groups. The siRNA-Notch1, γ-secretase inhibitor, and Notch1-siRNA + γ-secretase inhibitor groups were treated with Notch1 siRNA and/or γ-secretase inhibitor. Following treatment, cell proliferation was evaluated using a Cell Counting Kit-8. Tumor-related factors, including transforming growth factor (TGF)-β1, c-Myc and p53, were detected by reverse transcription-quantitative polymerase chain reaction and western blot analyses. BMSC osteogenic differentiation was induced and the cells were stained with alizarin red at 14 and 21 days. Alkaline phosphatase (AKP) activity was also evaluated. The siRNA-Notch1 and γ-secretase inhibitor both reduced BMSC proliferation and the expression of TGF-β1 and c-Myc and increased the expression of p53. Following the induction of osteogenesis and staining with alizarin red, the level of AKP was significantly higher in cells in the siRNA-Notch1 and γ-secretase inhibitor groups compared with that in the control group. It was found that Notch1 inhibition reduced proliferation and promoted the osteogenic differentiation of BMSCs.

NKAP alters tumor immune microenvironment and promotes glioma growth via Notch1 signaling

BackgroundGlioma is one of the most aggressive malignant brain tumors which is characterized with highly infiltrative growth and poor prognosis. NKAP (NF-κB activating protein) is a widely expressed 415-amino acid nuclear protein that is overexpressed by gliomas, but its function in glioma was still unknown.MethodsCCK8 and EDU assay was used to examine the cell viability in vitro, and the xenograft models in nude mice were established to explore the roles of NAKP in vivo. The expressions of NKAP, Notch1 and SDF-1 were analyzed by immunofluorescence analysis. The expression of NKAP and Notch1 in glioma and normal human brain samples were analyzed by immunohistochemical analysis. In addition, CHIP, Gene chip, western blot, flow cytometry, immunofluorescence, ELISA and luciferase assay were used to investigate the internal connection between NKAP and Notch1.ResultsHere we showed that overexpression of NKAP in gliomas could promote tumor growth by contributing to a Notch1-dependent immune-suppressive tumor microenvironment. Downregulation of NKAP in gliomas had abrogated tumor growth and invasion in vitro and in vivo. Interestingly, compared to the control group, inhibiting NKAP set up obstacles to tumor-associated macrophage (TAM) polarization and recruitment by decreasing the secretion of SDF-1 and M-CSF. To identify the potential mechanisms involved, we performed RNA sequencing analysis and found that Notch1 appeared to positively correlate with the expression of NKAP. Furthermore, we proved that NKAP performed its function via directly binding to Notch1 promoter and trans-activating it. Notch1 inhibition could alleviate NKAP’s gliomagenesis effects.Conclusionthese observations suggest that NKAP promotes glioma growth by TAM chemoattraction through upregulation of Notch1 and this finding introduces the potential utility of NKAP inhibitors for glioma therapy.

Notch-1 Inhibition Promotes Immune Regulation in Transplantation Via Regulatory T Cell-Dependent Mechanisms.

Transplantation is the treatment of choice for many patients with end-stage organ disease. Despite advances in immunosuppression, long-term outcomes remain suboptimal, hampered by drug toxicity and immune-mediated injury, the leading cause of late graft loss. The development of therapies that promote regulation while suppressing effector immunity is imperative to improve graft survival and minimize conventional immunosuppression. Notch signaling is a highly conserved pathway pivotal to T-cell differentiation and function, rendering it a target of interest in efforts to manipulate T cell-mediated immunity. We investigated the pattern of Notch-1 expression in effector and regulatory T cells (Tregs) in both murine and human recipients of a solid-organ transplant. Using a selective human anti-Notch-1 antibody (aNotch-1), we examined the effect of Notch-1 receptor inhibition in full major histocompatibility complex-mismatch murine cardiac and lung transplant models, and in a humanized skin transplant model. On the basis of our findings, we further used a genetic approach to investigate the effect of selective Notch-1 inhibition in Tregs. We observed an increased proportion of Tregs expressing surface and intracellular (activated) Notch-1 in comparison with conventional T cells, both in mice with transplants and in the peripheral blood of patients with transplants. In the murine cardiac transplant model, peritransplant administration of aNotch-1 (days 0, 2, 4, 6, 8, and 10) significantly prolonged allograft survival in comparison with immunoglobulin G-treated controls. Similarly, aNotch-1 treatment improved both histological and functional outcomes in the murine lung transplant model. The use of aNotch-1 resulted in a reduced proportion of both splenic and intragraft conventional T cells, while increasing the proportion of Tregs. Furthermore, Tregs isolated from aNotch-1-treated mice showed enhanced suppressive function on a per-cell basis, confirmed with selective Notch-1 deletion in Tregs (Foxp3EGFPCreNotch1fl/fl). Notch-1 blockade inhibited the mammalian target of rapamycin pathway and increased the phosphorylation of STAT5 (signal transducer and activator of transcription 5) in murine Tregs. Notch-1low Tregs isolated from human peripheral blood exhibited more potent suppressive capacity than Notch-1high Tregs. Last, the combination of aNotch-1 with costimulation blockade induced long-term tolerance in a cardiac transplant model, and this tolerance was dependent on CTLA-4 (cytotoxic T-lymphocyte-associated antigen-4) signaling. Our data reveal a promising, clinically relevant approach for immune modulation in transplantation by selectively targeting Notch-1.

Abstract 302: The combined inhibition of Plk1 and Notch1 results in a synergistic anti-proliferative response in human melanoma cells

Abstract Melanoma is the most aggressive form of skin cancer, and its incidence and mortality have been increasing over the past thirty years. Therefore, additional therapeutic strategies are needed for an effective management of this neoplasm. We previously demonstrated that polo-like kinase 1 (Plk1, a serine/threonine kinase involved in mitotic regulation) and Notch1 (a type I transmembrane protein deciding cell fate during development) are strongly correlated in melanoma (Cancer Res 2018; 78 (13 Suppl): Abstract nr 2530). In addition, a number of studies from our lab and elsewhere have suggested the important functions of Plk1- as well as Notch- signaling in melanoma progression. Based on these observations, in this study, we determined the potential efficacy of a combined inhibition of Plk1 and Notch1 against melanoma cells. Employing Plk1 inhibitor volasertib (BI6727) and Notch1 inhibitor MK-0752, we determined the effects of concomitant targeting of these pathways in A375 (wild type TP53) and SK-MEL-2 (mutant TP53) human melanoma cell lines. Employing RealTime-Glo and trypan blue exclusion assays, we found that volasertib (10 and 20 nM) and MK-0752 (50 and 100 μM) resulted in a significant decrease (60-80%) in viability and growth of human melanoma cells A375 and SK-MEL-2. The Combination Index (CI), as calculated using the Chou-Talalay theorem, was less than 1 when volasertib at 10 nM was given with MK-0752 at 50 and 100 μM, indicating a synergism between these two drugs. As a result of the synergism, the melanoma cells treated with combined drugs showed decreased colony formation ability compared to individual drugs. In addition, the combined treatment also resulted in enhanced apoptosis of melanoma cells. Interestingly, cell cycle analysis by flow cytometry showed that combined inhibition of Plk1 and Notch1 caused a G2-M phase arrest of melanoma cells. Furthermore, our data demonstrated that volasertib and MK-0752 combination caused a marked increase of cleaved PARP (poly ADP ribose polymerase) and cleaved caspase-3 in both melanoma cell lines, as well as increased expression of tumor suppressor protein TP53 and its target p21 (CDKN1A) in A375 melanoma cells. Taken together, our data demonstrated that a combined targeting of Plk1- and Notch1- signaling pathways imparts a synergistic anti-proliferative response against melanoma. However, additional experiments are needed i) to validate our in vitro data to in vivo situations in human relevant melanoma models, and ii) to define the interactive mechanism(s) of the observed synergistic response. Citation Format: Shengqin Su, Gagan Chhabra, Chandra K. Singh, Mary A. Ndiaye, Nihal Ahmad. The combined inhibition of Plk1 and Notch1 results in a synergistic anti-proliferative response in human melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 302.

Abstract 1291: Notch2 inhibition as a therapeutic intervention in osteosarcoma

Abstract Notch signaling pathway is a mediator of cell differentiation and is critical for normal bone development. Four functional Notch ligands DLL1 and DLL4, and JAG1 and JAG2 showed various levels of affinity for Notch1-4 receptors. Dose dependent Notch signaling activation or blocking is attributed to anchored or soluble form of Notch ligands binding. Interestingly, Notch pathway is shown to play a dual role, either oncogenic or tumor suppressive, depending on signal dose and tissue-context. To understand Notch signaling activities in osteosarcoma (OS), we first studied the cell surface levels of Notch receptor expression in OS cell lines and patient derived xenograft (pdx) models. Cytometry based screening of NOTCH1-4 receptors showed NOTCH2 as a predominant surface expressed protein in most of the samples. Interestingly, the canonical Notch target genes HES1 and DTX1 were higher (~5-fold) in OS cells compared to mesenchymal cells (MSC). Subsequently, we evaluated selected Notch pathway gene expression in 48 patients with recurrent/metastatic OS by RNAseq analysis. High expression of Notch pathway-related genes was seen in a subset of patients. NOTCH2 expression was comparable between primary and metastatic OS specimens, highest as compared to other Notch receptors and was significantly higher than normal tissue. Thus we aimed to elucidate soluble Notch ligands mediated blocking of Notch signaling using immunoglobulin-G bound DLL-1, -3, and -4 and JAG-1, and -2 in in vitro studies using OS cells. Soluble DLL1-Fc treatment increased OS cell death as compared to the other Notch ligands as well as control-Fc cells. Addition of soluble DLL1 resulted in the decreased expression of Notch downstream genes such as HES1, DTX1 and HEY1. Taken together, these findings identify soluble DLL1 as a potent Notch pathway inhibitor in OS in vitro and support further exploration of NOTCH2 as a potential therapeutic target in OS. Citation Format: Sankaranarayanan Kannan, John A. Livingston, Michael Roth, Jonathan Benjamin, Yifei Wang, Zhongting Zhang, Wendong Zhang, Chia-Chin Wu, Hannah Beird, Andrew Futreal, Richard Gorlick. Notch2 inhibition as a therapeutic intervention in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1291.

Abstract 2834: Bacteroides fragilis: A potential pathogen orchestrating EMT and stemness in breast epithelial cells via concomitant activation of Notch and βcatenin axes

Abstract Background: Last decade established significant contributions of microbiome to organ specific cancers. A few recent studies suggested the existence of distinct breast microbiota and a shift in microbial community composition in diseased breast compared to normal breast. However, their functional impact and underlying mechanisms are unknown. Present study was designed to examine the contribution of pro-carcinogenic bacteria in breast cancer initiation, growth and progression. Results: Utilizing extensive data mining and metagenomic analyses, we discovered presence of toxin producing Bacteriodes fragilis in malignant breast. B. fragilis is known for its potential to initiate and/or promote colon cancer. Its pathogenicity has been attributed to its unique toxin BFT. Mice infected with B. fragilis exhibited significant circulating BFT and distinct morphological alterations in mammary gland. While no changes were observed in cell growth and clonogenicity upon BFT treatment, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells were observed. BFT-treated cells displayed acquisition of fibroblast-like appearance and increased formation of pseudopodia/microtentacles emanating from the cell membrane along with molecular markers of epithelial-to-mesenchymal transition. Decreased expression of epithelial marker, E-cadherin along with elevated levels of mesenchymal markers, N-cadherin and vimentin were observed. BFT also increased expression of EMT-related transcription factors, Snail, Slug and Twist. BFT-treated cells attained stem cell-like phenotype exhibiting an increased ability to form secondary and tertiary mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2). Mechanistic studies showed BFT induced expression and nuclear translocation of cleaved NOTCH1 and βcatenin resulting in activation of downstream targets. Inhibition of Notch1 and βcatenin using γ-secretase inhibitor and ICG001 successfully inhibited functional effects of BFT. Further, BFT-pretreated MCF7 cells exhibit increased tumor growth and form multifocal tumors in mice. MCF10A-KRas cells, pretreated with BFT, also showed increased tumor progression and multifocal tumors in mice. In vivo limiting dilution assay using breast tumors from BFT-pretreated MCF7 cells exhibited a striking increase in tumor-initiating cells. Follow-up analyses of these tumors demonstrated increased migratory, invasive, and mammospheres-forming behavior confirming that brief BFT exposure elicits long-term molecular changes. Conclusion: Collectively, these findings present the first in vitro and in vivo evidence to show that Bacteriodes fragilis Toxin induces EMT, invasion/migration and stem cell-like phenotype and leads to concomitant activation of Notch and βcatenin axes. Citation Format: Sheetal Parida, Shaoguang Wu, Nethaji Muniraj, Sumit Siddharth, Arumugam Nagaligam, Christina Hum, Panagiotis Mistriotis, Konstantinos Konstantopoulos, Cynthia L. Sears, Dipali Sharma. Bacteroides fragilis: A potential pathogen orchestrating EMT and stemness in breast epithelial cells via concomitant activation of Notch and βcatenin axes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2834.

Abstract 103: Disruption of Notch Signaling targeted to the myeloma bone marrow microenvironment simultaneously inhibits tumor growth and prevents bone loss without inducing gut toxicity

Abstract Communication between myeloma (MM) cells and cells of the bone marrow via Notch signaling promotes tumor growth/survival and stimulates bone resorption. Systemic inhibition of Notch, using γ-secretase inhibitors (GSIs), decreases MM growth and reduces bone destruction, but the clinical use of GSIs is limited due to dose-limiting severe gut toxicity. To circumvent GSI side effects, we generated a bone specific Notch inhibitor (BT-GSI) by conjugating GSI-XII to a targeting molecule (BT) with high bone affinity using an acid hydrolyzable linker. In vitro, BT-GSI was inactive unless pre-incubated at low pH, and exhibited equal inhibition of Notch target genes in MM cells as unconjugated GSI. Ex vivo, BT-GSI decreased Notch expression and reduced MM growth in bone organ cultures that reproduce acidic conditions in the MM-bone microenvironment. In vivo, treatment with BT-GSI (5mg/kg/3x/wk, i.p.) for 2 wks decreased Notch signaling in bone more efficiently than unconjugated GSI (10mg/kg/5x/wk, i.p.) in naïve mice. In addition, BT-GSI increased cancellous bone mass (30%) and decreased bone resorption by 40%, without affecting bone formation. In contrast, these parameters remained unchanged by GSI. Next, we examined in vivo the impact of BT-GSI on MM growth and bone disease in a preclinical model of established MM. 8-wk-old immunodeficient mice were injected intratibially with 105 JJN3 human MM (hMM) cells or saline. hMM injected mice exhibited detectable serum levels of the tumor biomarker human K-light chain (40 ng/mL) and visible osteolytic disease (osteolytic area 1.7 mm2) 3 wks after hMM inoculation. Then, hMM-injected mice were randomized based on tumor levels to two subgroups to receive either BT-GSI (10mg/kg/3x/wk) or vehicle (DMSO) for 3 wks. Saline-injected mice received vehicle injections. BT-GSI selectively decreased Notch gene expression in bone, but had no effect in the brain or gut. Further, BT-GSI did not increase the expression of Adipsin in the gut, a biomarker of gut toxicity, nor showed evidence of gut toxicity at necropsy. Mice treated with BT-GSI exhibited a 45% decrease in tumor burden (168 vs 254 ng/mL human K-light chain) and 50% less osteolytic area compared to vehicle treated mice bearing hMM (4.4 vs 10.2 mm2). Moreover, BT-GSI decreased serum CTX by 30%, but did not affect serum P1NP. Importantly, equimolar administration of the unconjugated BT molecule did not alter MM growth nor prevented bone loss in mice with established MM. In conclusion, these results show that bone-targeted Notch inhibition reduces MM growth and preserves bone mass in mice with established MM. Because BT-GSI shows bone specific Notch inhibition and lacks gut toxicity, it should circumvent the deleterious side effects that limit GSI use in patients. Thus, BT-GSI is a promising approach to inhibit MM growth and to prevent bone loss in MM patients. Citation Format: Adam J. Ferrari, Kevin McAndrews, Jessica H. Nelson, James T. Bell, Venkatesan Srinivasan, Frank H. Ebetino, Robert K. Boeckman Jr, G. David Roodman, Teresita Bellido, Jesus Delgado-Calle. Disruption of Notch Signaling targeted to the myeloma bone marrow microenvironment simultaneously inhibits tumor growth and prevents bone loss without inducing gut toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 103.

Abstract 859: Basal Notch4 activation is a druggable marker of aggressiveness in a panel of human cancer cell lines

Abstract Background: Notch pathway has been involved in cell differentiation, proliferation, apoptosis, angiogenesis and drug resistance, as well as in epithelial-to-mesenchymal transition. Notch pathway activation leads to a proteolytic cleavage, releasing the Notch intracellular domain (NICD), which translocates to the nucleus and activates target genes, such as HES1 (interacting with Notch for maintenance of stem cells and progenitor phenotype). Our team has demonstrated the involvement of PlGF/VEGFR1/Notch4 axis in the angiogenesis of hepatocellular carcinoma, but what about the role of Notch in other cancer types? The aim of this work is to characterize the basal activation and the role of Notch4 in a panel of human cancer cell lines. Materials and Methods: We characterized a panel of 8 pancreatic (PDAC), 8 head and neck (H&N), 5 colorectal (CRC), 5 cholangiocarcinoma (CK), and 10 hepatocellular (HCC) human carcinoma cell lines for intracellular domain of the notch protein 4 (NICD4), HES1, E-cadherin, and Vimentin expression by Western Blot. In each tumor type, cell lines with high and low Notch4 activation were selected to assess basal cell proliferation and migration, using MTT and wound-healing assay, respectively. In high versus low Notch4 cell lines, we assessed NUMB expression (involved in NICD proteasomal degradation) and the effect of a Notch inhibitor, PF- 03084014. Results: Notch4 activation assessed by NICD4 expression was observed in 6 out of 8 (6/8) PDAC, 7/8 H&N, 2/5 CRC, 4/5 CK, and 5/10 HCC cell lines. In PDAC cells, Notch4 activation was correlated with high Vimentin expression. In PDAC-, H&N-, CRC-, and CK-low NICD4 cells, we observed an overexpression of NUMB, suggesting a correlation between low Notch4 activation and proteasomal degradation. In most tumor types, Notch4 activation was associated with an increased proliferation rate and basal migration. PF-03084014 had pronounced antiproliferative effects in high NICD4 cells in comparison to low NICD4 cells, except for HCC. In H&N cells, PF-03084014 displayed no effect on Notch4 activation. We also observed a decrease in HES1 expression (significant in high NICD4 cells) and an increased in AKT phosphorylation (significant in low NICD4 cells). We will further analyze the effect of PF-03084014 on cell signaling and migration in other tumor types to display the results at the conference. Conclusions: In this study, we demonstrated a correlation between high Notch4 basal activation and increased proliferation/migration in a panel of human cancer cell lines, as well as higher sensitivity to the Notch inhibitor PF-03084014. In highly sensitive H&N cell lines, PF-03084014 inhibited Notch signaling pathway, whereas we observed an activation of AKT survival pathway in the low-sensitive ones. Since Notch inhibition is an interesting topic for antitumor therapy, this study could help to select tumor types that could be good candidate for Notch inhibition in the clinic. Citation Format: Lucile Astorgues-Xerri, Matthieu Martinet, Eric Raymond, Sandrine Faivre, Annemilaï Tijeras-Raballand. Basal Notch4 activation is a druggable marker of aggressiveness in a panel of human cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 859.

Abstract 3439: Sulindac sulfide as a gamma secretase modifier to target triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is defined as pathologically negative for estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 amplification (HER2-). TNBCs are a heterogeneous group of clinically aggressive breast cancers with high risk of recurrence and metastasis, but the current treatment options remain limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, Including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC. However the success of GSIs in clinical trials is limited by their intestinal toxicity and adverse immunological effects. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSIs. SS has documented Gamma Secretase Modifier (GSM) activity, in addition to cyclo-oxygenase (COX) inhibition. We confirmed that SS inhibits Notch1 cleavage in TNBC cells, but not in murine T-cells. SS significantly inhibited mammospheres growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In C0321 tumors, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors. SS caused an increase in intra-tumoral CD11c+ dendritic cells, but decreased CD4 cells, which in this model are largely PD-1 positive (exhausted). CD8 cells were modestly increased. SS did not affect the numbers of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. RNA-Sequencing of SS-treated tumors revealed significant reduction of CXCL14, EGR1, HOXC6, MAGI2, NCAM1, APOE, CLU (a Wnt target), DTX4 (an E3-ligase positive regulator of Notch activation), and TGFB3 genes and upregulation of CCL17, EPCAM, FABP4, C4A, LTF, ZBTB16, INADL, and FGFR2 genes. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be significantly easier and more cost-effective than developing unproven investigational agents. Citation Format: Fokhrul Hossain, Deniz A Ucar, Samarpan Majumder, Margarite Matossian, Keli Xu, Yong Ran, Lisa Minter, Yaguang Xi, Matthew Burow, Todd Golde, Barbara Osborne, Lucio Miele. Sulindac sulfide as a gamma secretase modifier to target triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3439.

Abstract 2695: Direct targeting of NOTCH transcription complex by a novel small molecule CB-103 circumvents dose-limiting toxicities associated with pan-NOTCH inhibitors

Abstract NOTCH signalling is a key development pathway whose aberrant activation plays an onco-driver role in human cancers. In human tumors the NOTCH pathway can be activated by various genetic lesions such as over expression of ligands/receptors, GOF mutations in NOTCH receptors, chromosomal translocations, or loss-of-function mutations in the negative regulators of the pathway. Activation of NOTCH due to above mentioned mechanisms can be addressed in part using blocking antibodies against NOTCH ligands/receptors or small molecule inhibitors of the gamma secretase enzyme (GSIs). In addition to their limitations in targeting tumors harbouring chromosomal translocations in NOTCH receptors, the use of specific blocking antibodies against NOTCH receptors/ligands allows targeting of only a narrow spectrum of NOTCH positive tumors. On the other hand, GSIs act as pan-NOTCH inhibitors and thereby are able to target human tumors positive for NOTCH1-NOTCH4 receptors. However, clinical development of these pan-NOTCH inhibitors is hindered due to dose limiting toxicities (DLTs) associated with GSIs. The main DLTs associated with GSIs have been diarrhoea and vomiting due induction of goblet cell metaplasia in intestine, where NOTCH is known to regulate proliferation and differentiation of intestinal stem cells. A sustained blockage of NOTCH signalling leads to an inhibition of stem cell proliferation and differentiation of stem cells into mucous producing goblet cells. The effect on stem cell proliferation and differentiation is mediated via a subset of specific NOTCH target genes. Effect on proliferation is mostly driven by downstream target genes such as HES1, 3, and 5, cMYC and BMI1. The effect on stem cell differentiation is predominantly mediated by repression of MATH1. Therefore, MATH1 upregulation upon NOTCH inhibition is a pre-requisite to induce goblet cell metaplasia. Given the role of NOTCH signalling in human cancers and DLTs associated with 1st and 2nd generation NOTCH targeting agents (GSIs and blocking Abs), there is a need to develop novel pan-NOTCH inhibitors able to circumvent these DLTs. We have previously reported discovery and development of a novel class of pan-NOTCH inhibitors that blocks signalling by directly targeting the NOTCH transcription complex. Here we present further in vivo characterization of the development candidate CB-103. A comprehensive in vitro study demonstrated that CB-103 acts as a pan-NOTCH inhibitor. Furthermore, in vitro and in vivo pharmacological studies show that CB-103 circumvents DLTs, namely goblet cell metaplasia, associated with GSIs, due to the novel mode of action. We present molecular evidence that due to differential regulation of downstream target genes, CB-103 fully engages the NOTCH pathway in intestine and spares MATH1 gene repression and thereby does not cause goblet cell metaplasia. Citation Format: Rajwinder Lehal, Jelena Zaric, Michele Vigolo, Charlotte Urech, Maximilien Murone, Freddy Radtke. Direct targeting of NOTCH transcription complex by a novel small molecule CB-103 circumvents dose-limiting toxicities associated with pan-NOTCH inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2695.

Abstract 3468: Aberrant activation of gli1 and notch1 contributes to racial disparity in triple negative breast cancer progression

Abstract Background and Aim: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor and HER2 expression. Mortality from TNBC is significantly higher in African American (AA) women compared to European American (EA) women (5-year relative survival of 14% for AA compared to 36% for EA). Irrespective of stage at diagnosis, AA-TNBC is aggressive with higher metastasis and poorer survival than EA-TNBC; hence, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall this study aims to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential in comparison to EA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. AA-TNBC cells also exhibited higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population. To decipher the molecular mechanism underlying these functional differences, we analyzed RNA sequencing data of multiple AA and EA TNBC cell lines for self-renewal pathways and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC (Pearson coefficient = 0.308303) with a negative correlation in EA-TNBC (Pearson coefficient = -0.06695). Immunoblot analyses showed increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, NICD, Hes1 and FOXM1) in AA-TNBC compared to EA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to EA-TNBC cells. We observed that GLI and NICD co-localize in AA-TNBC cells and their interaction was confirmed using co-immunoprecipitation assays. High expression of GLI1 and Notch1 correlated with poor overall survival in TNBC patients. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and Carboplatin) effectively inhibited AA TNBC growth and progression in mice; proliferation, migration and invasion of ex vivo tumor cells and downregulated CD44+/CD49f+ and ALDH1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Citation Format: Sumit Siddharth, Nethaji Muniraj, Sheetal Parida, Arumugam Nagalingam, Dipali Sharma. Aberrant activation of gli1 and notch1 contributes to racial disparity in triple negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3468.

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.

Abstract 4837: Role of Notch signaling in human mucoepidermoid carcinoma and combined targeting of Notch and EGFR signaling as an anti-cancer strategy

Abstract Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy and currently no targeted therapy is available for MEC patients. MEC is characterized by a unique chromosomal translocation t(11;19)(q14-21;p12-13) that generates the CRTC1-MAML2 fusion. Our understanding of CRTC1-MAML2-mediated tumorigenesis and maintenance remains limited. MEC is considered a disease of stem/progenitor cells; however, the regulation of human MEC stem/progenitor cells has not yet been characterized. In this study, we focused on Notch signaling, a pathway important in regulating normal and cancerous stem cells, and investigated its regulatory role in human MEC cancer stem/progenitor cells as well as its potential therapeutic potential. First, we determined the status of Notch signaling activation in MEC cells. Western blotting showed the presence of cleaved active NOTCH1 and the expression of Notch downstream target gene HES1 in human CRTC1-MAML2 fusion-positive MEC cells, indicative of active Notch signaling in MEC. Second, we utilized two approaches of blocking endogenous Notch signaling in human MEC cells in vitro and in vivo, i.e. dnMAML1, a pan-Notch inhibitor that blocks the formation of the Notch transcriptional activation complex, and dibenzazepine (DBZ), a γ-secretase inhibitor that interferes with Notch receptor processing. We observed that Notch signaling inhibition had no effect on the proliferation of bulk MEC cells, but significantly reduced oncosphere forming capacity and ALDH-positive population in human MEC in vitro, suggesting that Notch signaling contributes to the maintenance of MEC stem/progenitor cells. Further, Notch signaling inhibition significantly reduced the growth of human MEC xenografts, indicating an essential role of Notch signaling in MEC growth in vivo. Finally, since we previously showed that the CRTC1-MAML2-induced autocrine AREG-EGFR signaling was required for the growth and survival of human MEC cells, we reasoned that simultaneous targeting of Notch and EGFR signaling will eliminate cancer stem and non-stem cell populations, likely improving therapeutic efficacy. Indeed, concurrent inhibition of Notch and EGFR signaling via respective DBZ and Erlotinib synergistically blocked the colony and oncosphere formation capacity of human MEC cells in vitro and in vivo. Collectively, our data demonstrate that Notch signaling is a critical regulator for maintaining MEC cancer-initiating cells and that the combination strategy targeting Notch and EGFR signaling is a potentially effective approach for MEC treatment. Citation Format: Wei Ni, Zirong Chen, Xin Zhou, Rongqiang Yang, Frederic Kaye, Lizi Wu. Role of Notch signaling in human mucoepidermoid carcinoma and combined targeting of Notch and EGFR signaling as an anti-cancer strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4837.

Abstract 4885: AL101 mediated tumor inhibition in Notch mutated ACC PDX models

Abstract Goal: To evaluate Notch inhibitor monotherapy and combination (combo) therapy in Adenoid Cystic Carcinoma (ACC) Patient Derived Xenograft (PDX) models with and without activating Notch mutations (mt). ACC is a rare salivary gland malignancy with no standard of care. Chemotherapy resistance limits treatment to surgery/radiation and ~60% of patients (pts) will recur. ~22% of ACC pts have Notch activating mutations that are associated with a distinct phenotype, aggressive disease and poor prognosis. In a P1 solid tumor study, 2 ACC pts were treated with AL101, an investigational gamma secretase inhibitor (J Clin Oncol 36, 2018 abstract 2515). One pt with an NRR (Negative Regulatory Region) activating mutation had a prolonged partial response, the 2nd pt with a PEST mutation had stable disease. Both had an extended PK profile with a sustained pharmacodynamic response (>50% inhibition of HES1 in peripheral blood). These results prompted us to evaluate the effect of AL101 in tumors that harbor/lack Notch activating mutations. Design: 4 ACC PDX models were evaluated: ACCx9 (Notch1 mt: NRR activating mutation), ACCx11 (Notch1 mt, tandem duplication), ACCx6 (Notch1 wt), ACCx5M1 (Notch1 VUS, not predicted to be activating). Activated Notch1 (nuclear IHC stain) was confined to the Notch mt models. Tumors were implanted into 6-12 week old athymic nude female mice. Upon reaching 150-300 mm3 tumor volume, mice were randomized to treatment (N=5), or vehicle (N=10) arms. AL101 was dosed at 7.5 mg/kg po (days 1-4 of Q7D) as single agent or in combo with cisplatin (3 mg/kg ip; qw) or everolimus (10 mg/kg; po; qd). Results: Significant tumor growth inhibition (TGI) was seen with AL101 monotherapy compared to vehicle treatment in both Notch mt models (ACCx9; 110% TGI P<0.0001, and ACCx11; 78% TGI P=0.0441). AL101 had no significant effect on tumors lacking Notch activating mutations (ACCx6: 37% TGI P>0.99, ACCx5M1: 60% TGI P=0.26). Cisplatin and everolimus monotherapy had no significant effect on Notch mt models (cisplatin: ACCx9; 43% TGI P=0.98, ACCx11; 50% TGI P=0.99, everolimus: ACCx9; 51% TGI P=0.6, ACCx11; 54% TGI P=0.99). AL101 with cisplatin or everolimus had no further benefit in the ACCx9 model (both 106% TGI P=0.98 or P>0.99 respectively). The ACCx11 model had a non-significant benefit (104% TGI P=0.17 for cisplatin combo and 107% TGI P=0.18 for everolimus combo). Interestingly, the ACCx6 Notch wt model was sensitive to everolimus (75% TGI P=0.027) with no significant benefit of adding AL101. Conclusion: AL101 monotherapy had a significant anti-tumor effect in ACC PDX tumors with Notch activating mutations and lacked effectiveness in tumors lacking such mutations. Neither cisplatin nor everolimus monotherapy treatment had a significant effect in Notch mt models, alone or in combination with AL101. These data support the clinical development of AL101 as a targeted monotherapy for ACCs with Notch activating mutations. Citation Format: Renata ferrarotto*, Genia Alpert*, Udi Gluschnaider, Rami Rauch, Adi Mondshine, Oz Solomon, Bill Kramer, Evgeny Izumchenko, John Heymach, Andrea Vergara-Silva, Jon Aster, Matti Davis. AL101 mediated tumor inhibition in Notch mutated ACC PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4885.