Notch Transcriptional Complex Research Articles

Unraveling paralog-specific Notch signaling through thermodynamics of ternary complex formation and transcriptional activation of chimeric receptors.

Notch signaling in humans is mediated by four paralogous receptors that share conserved architectures and possess overlapping, yet non-redundant functions. The receptors share a canonical activation pathway wherein upon extracellular ligand binding, the Notch intracellular domain (NICD) is cleaved from the membrane and translocates to the nucleus where its N-terminal RBP-j-associated molecule (RAM) region and ankyrin repeat (ANK) domain bind transcription factor CSL and recruit co-activator Mastermind-like-1 (MAML1) to activate transcription. However, different paralogs can lead to distinct outcomes. To better understand paralog-specific differences in Notch signaling, we performed a thermodynamic analysis of the Notch transcriptional activation complexes for all four Notch paralogs using isothermal titration calorimetry. Using chimeric constructs, we find that the RAM region is the primary determinant of stability of binary RAMANK:CSL complexes, and that the ANK regions are largely the determinants of MAML1 binding to pre-formed RAMANK:CSL complexes. Free energies of these binding reactions (ΔGRA and ΔGMAML) vary among the four Notch paralogs, although variations for Notch2, 3, and 4 offset in the free energy of the ternary complex (ΔGTC, where ΔGTC = ΔGRA + ΔGMAML). To probe how these affinity differences affect Notch signaling, we performed transcriptional activation assays with the paralogous and chimeric NICDs, and analyzed the results with an independent multiplicative model that quantifies contributions of the paralogous RAM, ANK, and C-terminal regions (CTR) to activation. This analysis shows that transcription activation correlates with ΔGTC, but that activation is further modified by CTR identity in a paralog-specific way.

The structure, bindingand function of a Notch transcription complex involving RBPJ and the epigenetic reader protein L3MBTL3.

The Notch pathway transmits signals between neighboring cells to elicit downstream transcriptional programs. Notch is a major regulator of cell fate specification, proliferation, and apoptosis, such that aberrant signaling leads to a pleiotropy of human diseases, including developmental disorders and cancers. The pathway signals through the transcription factor CSL (RBPJ in mammals), which forms an activation complex with the intracellular domain of the Notch receptor and the coactivator Mastermind. CSL can also function as a transcriptional repressor by forming complexes with one of several different corepressor proteins, such as FHL1 or SHARP in mammals and Hairless in Drosophila. Recently, we identified L3MBTL3 as a bona fide RBPJ-binding corepressor that recruits the repressive lysine demethylase LSD1/KDM1A to Notch target genes. Here, we define the RBPJ-interacting domain of L3MBTL3 and report the 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA complex. The structure reveals that L3MBTL3 interacts with RBPJ via an unusual binding motif compared to other RBPJ binding partners, which we comprehensively analyze with a series of structure-based mutants. We also show that these disruptive mutations affect RBPJ and L3MBTL3 function in cells, providing further insights into Notch mediated transcriptional regulation.

Histone chaperone ASF1A accelerates chronic myeloid leukemia blast crisis by activating Notch signaling

The blast crisis (BC) is the final deadly phase of chronic myeloid leukemia (CML), which remains a major challenge in clinical management. However, the underlying molecular mechanism driving blastic transformation remains unclear. Here, we show that ASF1A, an essential activator, enhanced the transformation to CML-BC by mediating cell differentiation arrest. ASF1A expression was aberrantly increased in bone marrow samples from CML-BC patients compared with newly diagnosed CML-chronic phase (CP) patients. ASF1A inhibited cell differentiation and promoted CML development in vivo. Mechanistically, we identified ASF1A as a coactivator of the Notch transcriptional complex that induces H3K56ac modification in the promoter regions of Notch target genes, and subsequently enhanced RBPJ binding to these promoter regions, thereby enhancing Notch signaling activation to mediate differentiation arrest in CML cells. Thus, our work suggests that targeting ASF1A might represent a promising therapeutic approach and a biomarker to detect disease progression in CML patients.

Developing RNA‐Seq and ChIP‐Seq Pipelines on an Oxford Nanopore Technologies Platform to Study Notch1 Transcriptional Dynamics

Notch signaling plays a crucial role in cell development and proliferation. The Notch receptor is an inducible transcription factor found on the cell surface which is cleaved resulting in the release of the intracellular domain (NICD). The NICD enters the nucleus and complexes with CBF1 and Mastermind (MAM) to activate target genes. CBF1 recognizes the binding motif RTGRGAR (R=G or A), and this is referred to an NRE (Notch Response Element). Recent studies have shown that a paired CBF1 binding site is also important for target gene activation. While individual genes regulated by SPS are known, the extent to which an SPS vs individual NREs are responsible for Notch‐mediated transcription on a genomic level remains an active area of research. It is hypothesized that the majority of Notch‐mediated transcription occurs as a result of a dimeric Notch complex in the promoters or enhancers of transcribed genes. Recently, high‐throughput techniques have allowed the study of Notch complex assembly and their effects on transcription. Here, we use an Oxford Nanopore Platform was used.To identify genes regulated by Notch transcriptional complexes, RNA‐Seq was performed. Total RNA was isolated from SUPT1 cells treated with 5 µM of the Notch inhibitor DAPT for 24 hours, or DMSO as a control. Poly‐A RNA was purified using an oligo‐dT column and double‐stranded cDNA generated. PCR adapters were nonspecifically ligated to cDNA and amplified to increase library yield. Sequencing was performed using an Oxford Nanopore Minion, and results were mapped to the human reference genome to identify genes significantly downregulated when Notch was inhibited. To identify NREs and SPS in the regulatory elements of these genes, ChIP‐Seq was performed for Notch. ChIP was performed in DAPT and DMSO treated cells, and ChIP DNA used to prepare a sequencing library as above. Sequenced fragments were aligned to a known regulatory elements dataset, and genes with significant Notch binding identified.RNA‐Seq data shows significant downregulation of known Notch targets genes, such as Hes1, Hey1, and Hes4. Sequenced mRNA fragments, however, averaged 1kb suggesting size selectivity during the purification or amplification steps of library preparation. Additionally, underrepresentation of other Notch known targets such as Hes5 may suggest that computational stringency may have to be adjusted. We are currently pursuing ChIP‐Seq experiments that will be presented at the conference to identify the locations of SPS in the genome. Identified SPS in regulatory elements will then be correlated with the genes they activate, and the downstream effects on transcription analyzed. It is expected that a large number of genes that are inactivated under Notch inhibition conditions will exhibit a SPS in a corresponding regulatory element.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

MTG16 Variants Provide Context to Notch Signaling to Direct Leukemic Phenotype

Notch signaling guides hematopoietic stem and progenitor cells (HS/PCs) toward distinct cell fates and when dysfunctional, contributes to development of hematopoietic malignancies. Normally, Notch signaling favors T-cell fate and causes attenuated expression of myeloid-specific genes. In contrast, Notch pathway inactivation causes HS/PCs to diverge disproportionately toward a myeloid fate and is accompanied by T-lymphopenia. Mutations that constitutively activate Notch signaling trigger T-cell leukemia, while Notch inactivation is associated with myeloid leukemias. This suggests that Notch-regulated cell fate decisions are context dependent and that determinants downstream of Notch can influence Notch-dependent phenotypes in development and disease.Myeloid Translocation Gene (MTG) 16 binds the intracellular domain of Notch (NICD) and this interaction is necessary to establish CD4+/CD8+ (double positive-DP) T-cell fate. Mtg16 -/- progenitors give rise to GR1+/Mac1+ progeny in response to Notch activation. In ex vivo assays, normal DP T-cell development can be restored to Mtg16 -/- progenitors by enforced expression of MTG16, but not an MTG16 derivative which lacks NICD binding and mimics a naturally occurring Mtg16 splice variant (MTG16c). These findings suggest the status of expressed Mtg16 isoforms can determine lineage allocation in response to Notch signaling and intimates that Notch signaling could trigger alternate phenotypes depending upon the context in which it is activated.To assess impact of the NICD-MTG16 interaction as a determinant of malignant phenotype in the context of constitutive Notch signaling, we created bicistronic retroviral constructs whose NICD and MTG16 expression cassettes were separated by the “self-cleaving” 2A peptide of Foot and Mouth disease virus (FMDV). In each transduced progenitor cell, this insures concurrent stoichiometric expression of NICD and the chosen MTG16 derivative (wild type or NICD binding-deficient). Transduced Mtg16 -/- HS/PCs were then transplanted into irradiated, syngeneic recipients and leukemic phenotype established by flow cytometry. Mice transplanted with Mtg16 -/- cells transduced with vector only fail to reconstitute hematopoiesis in the recipient bone marrow due to a previously described HS/PC defect. Instead, these mice succumb to marrow failure by day 18 post-transplant. However, enforced expression of NICD alone enables survival of Mtg16 -/- progenitors, reconstitution of hematopoiesis and development of T-cell leukemia from transplanted donor elements. This same phenotype was observed when NICD was co-expressed with MTG16, indicating MTG16 is required for normal T-lymphopoiesis but is dispensible for NICD-driven T-cell leukemia. Notably, when Mtg16 -/- progenitors are transduced with NICD and a NICD binding deficient derivative of MTG16, recipients develop T-myeloid bilineal leukemia. This finding suggests that integration of NICD into transcriptional regulatory complexes involving MTG16 can alter the features of leukemic blasts along a continuum from lymphoid to myeloid phenotypes. To gain additional insights, we characterized promoter occupancy and Notch transcription complex (NTC) composition upon expression of MTG16 and its NICD binding deficient derivative. We find both MTG16 forms can exist in the same transcriptional complexes via their NHR2 oligomerization domains and can co-occupy the Notch-responsive Hes1 promoter. Given that NICD-MTG16 binding disrupts interactions between MTG16, histone deacetylases (HDACs) and other co-repressors, these data suggest that leukemic phenotype arising from constitutive Notch signaling may be influenced by combinatorial diversity among MTG16 splice variants and differential integration of the NICD into the NTC. DisclosuresNo relevant conflicts of interest to declare.

Notch family members follow stringent requirements for intracellular domain dimerization at sequence-paired sites.

Notch signaling is essential for multicellular life, regulating core functions such as cellular identity, differentiation, and fate. These processes require highly sensitive systems to avoid going awry, and one such regulatory mechanism is through Notch intracellular domain dimerization. Select Notch target genes contain sequence-paired sites (SPS); motifs in which two Notch transcriptional activation complexes can bind and interact through Notch's ankyrin domain, resulting in enhanced transcriptional activation. This mechanism has been mostly studied through Notch1, and to date, the abilities of the other Notch family members have been left unexplored. Through the utilization of minimalized, SPS-driven luciferase assays, we were able to test the functional capacity of Notch dimers. Here we show that the Notch 2 and 3 NICDs also exhibit dimerization-induced signaling, following the same stringent requirements as seen with Notch1. Furthermore, our data suggested that Notch4 may also exhibit dimerization-induced signaling, although the amino acids required for Notch4 NICD dimerization appear to be different than those required for Notch 1, 2, and 3 NICD dimerization. Interestingly, we identified a mechanical difference between canonical and cryptic SPSs, leading to differences in their dimerization-induced regulation. Finally, we profiled the Notch family members' SPS gap distance preferences and found that they all prefer a 16-nucleotide gap, with little room for variation. In summary, this work highlights the potent and highly specific nature of Notch dimerization and refines the scope of this regulatory function.

Notch Inhibition in Cancer: Challenges and Opportunities.

Notch is a key oncogenic pathway in several human cancers and to date, no targeted treatment of Notch activated cancers is available to patients. Therapeutic targeting of Notch has been an unresolved challenge due to severe on-target dose limiting toxicities associated with pan-Notch inhibition by either γ-secretase inhibitors or receptor/ligand targeting MAbs. At Cellestia Biotech, we have identified novel series of small molecule inhibitors of the Notch transcription complex. These molecules act as pan-Notch inhibitors and do not cause toxicities commonly associated with first- and second-generation Notch inhibitors currently tested in the clinic, thus providing a novel and unique opportunity to address a high unmet medical need. Our lead molecule, CB-103 is currently being investigated in Phase-1 dose escalation in cancer patients. Cellestia Biothech is further expanding its medicinal chemistry activities advancing the development of novel molecules for targeting transcription factors in cancer as well as non-cancer indications.

562P Target engagement and clinical safety of CB-103, a first-in-class small molecule inhibitor of the NOTCH transcription complex

562P Target engagement and clinical safety of CB-103, a first-in-class small molecule inhibitor of the NOTCH transcription complex

Pharmacological disruption of the Notch transcription factor complex

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule γ-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lymphoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrangements in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that interferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch-driven cancers.

Top Notch Targeting Strategies in Cancer: A Detailed Overview of Recent Insights and Current Perspectives.

Evolutionarily conserved Notch plays a critical role in embryonic development and cellular self-renewal. It has both tumour suppressor and oncogenic activity, the latter of which is widely described. Notch-activating mutations are associated with haematological malignancies and several solid tumours including breast, lung and adenoid cystic carcinoma. Moreover, upregulation of Notch receptors and ligands and aberrant Notch signalling is frequently observed in cancer. It is involved in cancer hallmarks including proliferation, survival, migration, angiogenesis, cancer stem cell renewal, metastasis and drug resistance. It is a key component of cell-to-cell interactions between cancer cells and cells of the tumour microenvironment, such as endothelial cells, immune cells and fibroblasts. Notch displays diverse crosstalk with many other oncogenic signalling pathways, and may drive acquired resistance to targeted therapies as well as resistance to standard chemo/radiation therapy. The past 10 years have seen the emergence of different classes of drugs therapeutically targeting Notch including receptor/ligand antibodies, gamma secretase inhibitors (GSI) and most recently, the development of Notch transcription complex inhibitors. It is an exciting time for Notch research with over 70 cancer clinical trials registered and the first-ever Phase III trial of a Notch GSI, nirogacestat, currently at the recruitment stage.

A Test to Quantify NOTCH Pathway Activity in T Cell Acute Lymphoblastic Leukemia Patients

Background The NOTCH signaling pathway is pivotal for various physiological processes including immune responses, and has been implicated in the pathogenesis in many diseases including T-cell acute lymphoblastic leukemia (T-ALL). Over 70% of T-ALL patient samples contain mutations in NOTCH1 and/or FBXW7 that result in the activation of the NOTCH pathway. Various targeted drugs are available that inhibit NOTCH signaling, but their effectiveness varies due to variable NOTCH pathway activities among individual patients. Moreover, patients' leukemic cells that lack these mutations may still require NOTCH signaling. A quick and robust quantification of NOTCH pathway activity in primary patient samples would identify patients who could benefit from NOTCH targeted treatment. Aims In primary human T-ALL samples, we aimed to determine the NOTCH pathway activity in relation to active, intracellular NOTCH1 (ICN1) levels and in relation to NOTCH1 and/or FBXW7 mutations. Additionally, we investigated whether the NOTCH pathway activity score is more accurate than a mutation-based activity prediction. Methods Our test to assess functional NOTCH pathway activity in various cell types was applied to primary human T-ALL samples. The NOTCH test infers a quantitative NOTCH pathway activity score from mRNA levels of conserved direct NOTCH target genes based on a Bayesian network model. This model describes the causal relation between up- or downregulation of NOTCH target genes and the presence of an active or inactive NOTCH transcription complex. The Bayesian model was calibrated on publically available Affymetrix U133 Plus2.0 microarray datasets of samples with an active or inactive NOTCH pathway. Following validation on multiple cell types and malignancies, we scored NOTCH pathway activation in our well-characterized cohort of 117 T-ALL patient samples and related it to clinical and biological parameters including outcome. Results The NOTCH pathway model was calibrated using a microarray dataset containing high-grade serous ovarian cancer-which has high NOTCH activity-and normal ovarian tissue samples that lack NOTCH activity. Validation of the test using datasets from primary cells and cell lines of various origins revealed that it measures the NOTCH activity status in different cellular contexts. In primary diagnostic T-ALL samples, we observed a significant relationship between NOTCH pathway activity scores and active, intracellular cleaved NOTCH1 (ICN1) protein levels and the presence of NOTCH1-activating mutations. We next scored NOTCH pathway activity over the four T-ALL subgroups ETP-ALL, TLX, Proliferative and TALLMO. The TLX subgroup had the highest NOTCH activity levels compared to the other subgroups, consistent with the high percentage of TLX cases with NOTCH1/FBXW7 mutations. Strikingly, the significance of the correlation between ICN1 levels and NOTCH pathway activity was mainly attributed to the strong NOTCH1-activating mutations that include NOTCH1 juxtamembrane domain mutations, or hetero-dimerization mutations combined with PEST domain or FBXW7 mutations. When assessing the event-free survival and relapse-free survival curves, we observed that patients with the lowest (lower than the 25th-percentile) NOTCH pathway activity scores had the shortest event-free survival compared to the others (p<0.05, log-rank test). Summary/Conclusion High NOTCH pathway activation was mostly present in-but not limited to-T-ALL samples harboring strong NOTCH1 mutations, indicating that additional mechanisms can activate NOTCH signaling. Combined with the observation that the measured NOTCH pathway activity relates to ICN1 protein levels, this indicates that the pathway activity score more accurately reflects NOTCH pathway activity than the predicted activity based on NOTCH1 mutations alone. Disclosures Holtzer: Philips Research: Employment. Verhaegh:Philips Research: Employment. van de Stolpe:The Netherlands: Employment; Eindhoven: Employment; Philips Research: Employment.

Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling.

Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.

Structural and mechanistic basis for CBP/P300 recruitment to the Notch transcription complex

Structural and mechanistic basis for CBP/P300 recruitment to the Notch transcription complex

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 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.

PACT increases mammalian embryonic neural stem cell properties by facilitating activation of the notch signaling pathway

The protein activator of protein kinase R (PKR) (PACT) is known to play important roles in PKR regulation and microRNA biogenesis. Based on the observation that PACT is specifically expressed in the ventricular zone (VZ) at the mid-neurogenic period, we examine the role of PACT in this embryonic neural stem cell niche. Here, we provide the first evidence that PACT increases neurosphere formation, as well as expression of Notch target genes and the neural stem cell marker Sox2 in primary neural stem cells in vitro. Consistently, introduction of PACT into the mouse embryonic brain in utero increased the fraction of cells localizing to the VZ. We also show that the PACT-enhanced stemness of neural stem cells is PKR-independent. At the molecular level, PACT was revealed to physically interact with C promoter binding factor 1 (CBF1) and dramatically strengthen the association between CBF1 and Notch intracellular domain (NICD), which indicates stabilization of the Notch transcriptional coactivation complex responsible for Notch target gene expression. Taken together, our study indicates that PACT is a novel transcriptional coactivator of the Notch pathway playing a pivotal role during mammalian brain development.

Notch1 activates angiogenic regulator Netrin4 in endothelial cells.

Netrin4 (NTN4) is a chemotropic factor that regulates angiogenesis. We found that endothelial expression of the activated, intracellular domain of Notch1 (NICD1), significantly up‐regulated NTN4 mRNA as well as intracellular NTN4 protein in both transgenic mice and cultured human umbilical vein endothelial cells (HUVECs). Notch1 activation also increased NTN4 secretion from HUVECs. We subsequently demonstrated that NICD1 bound to CSL (CBF1, Suppressor of Hairless, Lag‐1), a core component of Notch transcription complex, at the −53 element of the human NTN4 gene promoter. Loss of the −53 element compromised NICD1‐induced NTN4 expression. Our results suggest a conserved role for Notch signalling in transcriptional regulation of endothelial NTN4.

Abstract P4-07-05: A novel, first in class Notch transcriptional inhibitor, CB-103 has activity on luminal breast cancer stem cells in combination with fulvestrant

Abstract The Notch signaling pathway plays a central role in cellular differentiation, growth and stem cell maintenance. Expression and activation of Notch pathway receptors and ligands have differential outcomes depending on the tissue, localization and cell type. When Notch pathway is aberrantly activated by genetic lesions, it can be a major driver for Notch-dependent cancers and can cause resistance to standard of care treatment. We and others have shown that in Estrogen Receptor (ER)-positive breast cancers, estrogen deprivation caused by endocrine therapy results in Notch1 and Notch4 activation. In turn, Notch1 stimulated ER-dependent transcription in the absence of estrogen, causing endocrine resistance. Combinations of Notch inhibitors and endocrine therapy are effective in preclinical models of ER-positive breast cancer and have shown promising signals in early clinical trials. Cellestia's lead development candidate CB-103 is a small molecule, first-in-class, oral pan-Notch inhibitor. CB-103 selectively blocks Notch pathway activation-related gene transcription through binding to a Notch specific protein in the transcription factor complex. The blockade occurs by protein-protein interaction inhibition with a binding site critical for the assembly of the Notch transcription complex. This is a unique mode of action, which allows blocking Notch signaling regardless of the genetic lesions which have activated the pathway. We have performed mammosphere assays to test the potency and efficacy of this compound on stem cell ability to form sphere. We used two different doses of CB-103; either alone or in combination with a fixed dose of Fulvestrant (30nM), a SERD, in our mammosphere assays. Two different ER+ luminal,endocrine resistant cell lines were tested and compared with their parental controls. From our data, it's apparent that there is a synergistic effect when using CB-103 in combination with Fulvestrant. It's also evident that the efficacy of CB-103 is maximal maximizes at the lowest concentration tested in our assays. The combination was effective in 3 out of 4 models. However, the effect of CB-103 on MCF7-TAMR either as a single agent or in combination was not statistically significant. Cellestia has received regulatory approval to start clinical development with CB-103 in a first-in-human study Phase l – lla study investigating safety (Ph l) and preliminary single agent efficacy (Ph lla) of CB-103 in patients with advanced solid cancers and haematological malignancies. Our data support the notion of testing this agent in ER-positive breast cancer in combination with SERDs. Citation Format: Majumder S, Crabtree J, Hossain F, Murone M, Lehal R, Miele L. A novel, first in class Notch transcriptional inhibitor, CB-103 has activity on luminal breast cancer stem cells in combination with fulvestrant [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-05.

The role of DEAD-box RNA helicase p68 (DDX5) in the development and treatment of breast cancer.

RNA helicase p68 or DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 (DDX5) is a unique member of the highly conserved protein family, which is involved in a broad spectrum of biological processes, including transcription, translation, precursor messenger RNA processing or alternative splicing, and microRNA (miRNA) processing. It has been shown that p68 is necessary for cell growth and participates in the early development and maturation of some organs. Interestingly, p68 is a transcriptional coactivator of numerous oncogenic transcription factors, including nuclear factor-κβ (NF-κβ), estrogen receptor α (ERα), β-catenin, androgen receptor, Notch transcriptional activation complex, p53 and signal transducer, and activator of transcription 3 (STAT3). Recent studies on the role of p68 (DDX5) in multiple dysregulated cellular processes in various cancers and its abnormal expression indicate the importance of this factor in tumor development. Discussion of the precise role of p68 in cancer is complex and depends on the cellular microenvironment and interacting factors. In terms of the deregulated expression of p68 in breast cancer and the high prevalence of this cancer among women, it can be informative to review the precise function of this factor in the breast cancer. Therefore, an attempt will be made in this review to clarify the tumorigenic function of p68 in association with its targeting potential for the treatment of breast cancer.