The Membrane-Bound Notch Regulator Mnr Supports Notch Cleavage and Signaling Activity in Drosophila melanogaster

The Notch signaling pathway is pivotal to cellular differentiation. Activation of this pathway involves proteolysis of the Notch receptor and the release of the biologically active Notch intracellular domain, acting as a transcriptional co-activator of Notch target genes. While the regulation of Notch signaling dynamics at the level of ligand–receptor interaction, endocytosis, and transcriptional regulation has been well studied, little is known about factors influencing Notch cleavage. We identified EP555 as a suppressor of the Notch antagonist Hairless (H). EP555 drives expression of CG32521 encoding membrane-bound proteins, which we accordingly rename membrane-bound Notch regulator (mnr). Within the signal-receiving cell, upregulation of Mnr stimulates Notch receptor activation, whereas a knockdown reduces it, without apparent influence on ligand–receptor interaction. We provide evidence that Mnr plays a role in γ-secretase-mediated intramembrane cleavage of the Notch receptor. As revealed by a fly-eye-based reporter system, γ-secretase activity is stimulated by the overexpression of Mnr, and is inhibited by its knockdown. We conclude that Mnr proteins support Notch signaling activity by fostering the cleavage of the Notch receptor. With Mnr, we identified a membrane-bound factor directly augmenting Notch intra-membrane processing, thereby acting as a positive regulator of Notch signaling activity.

Abstract P329: Exosomes Derived From Podoplanin Positive Cells Alter The Cellular Composition Of Healthy Mouse Hearts

Fibrosis and blood hypoperfusion stimulated by paracrine signals enhances the ventricular dysfunctionafter myocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN) positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells could independentlyalter healthy heart physiology and structure. PDPN+ cells were isolated 2 days after MI, culture expandedand activated with TNFα and Angiotensin II. Exosomes derived from activated PDPN+cells conditionedmedia (PDPN+exo) were used in vitro for the treatment of mouse cardiac endothelial cells (mCECs) andmouse fibroblast (3T3) and in vivo for the treatment of healthy mouse hearts. In vitro, PDPN+exoinfluenced the phenotype of mCECs, stimulating their lineage into lymphatic endothelial cells andfacilitated fibroblasts transition to myofibroblast. Characterization of the protein content of PDPN+exoshowed high concentration of Notch receptors and γ-Secretase, suggesting these cellular transitions maydepend on exosome-mediated Notch translocation and cleavage. In fact, after exosomes treatmentcleaved notch (NICD) translocated in the nuclei of mCECs and 3T3 as early as 1h of treatment and eitherHes-1 or Hey-1, major transcription factors activated by NICD were enhanced within 2d of treatment.Using DAPT, a γSecretase inhibitor, notch cleavage was inhibited, and no phenotype switching in responseto exosome treatment was observed. In vivo, PDPN+exo were injected into the left ventricle of healthymouse hearts followed by boosters delivered by retro-orbital vein injection. Treated mice developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increase of the enddiastolic and systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. Podoplanin positive cellsrepresent 80% of the scar’s cells of a chronic infarcted myocardium and the specific exosomes cargo highlyinfluence the lineage of cardiac cells altering the biology of endothelial cells and fibroblasts which mayfacilitate adverse remodeling.

Noncanonical Notch signals have opposing roles during cardiac development

The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fash- ion, which is conserved in Drosophila, Xenopus, and mammals. How- ever, the noncanonical role remains poorly understood in in vivo pro- cesses. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, po- tentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibit- ing Notch cleavage, shown to increase noncanonical Notch activ- ity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage- specific roles during cardiac development.

Proteomics of resistance to Notch1 inhibition in acute lymphoblastic leukemia reveals targetable kinase signatures

Notch1 is a crucial oncogenic driver in T-cell acute lymphoblastic leukemia (T-ALL), making it an attractive therapeutic target. However, the success of targeted therapy using γ-secretase inhibitors (GSIs), small molecules blocking Notch cleavage and subsequent activation, has been limited due to development of resistance, thus restricting its clinical efficacy. Here, we systematically compare GSI resistant and sensitive cell states by quantitative mass spectrometry-based phosphoproteomics, using complementary models of resistance, including T-ALL patient-derived xenografts (PDX) models. Our datasets reveal common mechanisms of GSI resistance, including a distinct kinase signature that involves protein kinase C delta. We demonstrate that the PKC inhibitor sotrastaurin enhances the anti-leukemic activity of GSI in PDX models and completely abrogates the development of acquired GSI resistance in vitro. Overall, we highlight the potential of proteomics to dissect alterations in cellular signaling and identify druggable pathways in cancer.

ACCURACY: phase (P) 2 trial of AL101, a pan-Notch inhibitor, in patients (pts) with recurrent/metastatic (R/M) adenoid cystic carcinoma (ACC) with Notch activating mutations (Notchact mut).

TPS6098 Background: Notch signaling plays a key role in tumorigenesis. Notch cleavage by γ-secretase frees the Notch intracellular domain, which promotes the expression of target genes involved in cancer. AL101, a small molecule, is a γ-secretase inhibitor that potently inhibits Notch1-4, resulting in robust antitumor activity in vivo (PMID 26005526), including ACC xenograft models with Notchact mut (Ferrarotto, AACR 2019, Abstr 4885). Three P1 trials tested AL101 as monotherapy or in combination regimens in > 200 solid/hematologic cancer pts. In the P1 trial of AL101 monotherapy, conducted in 94 pts with advanced/metastatic solid tumors refractory to standard therapies (Tx), AL101 was generally well tolerated, with manageable AEs, and the recommended P2 dose was 4 mg IV once weekly (QW; El-Khoueiry, ASCO 2018, Abstr 2515). 4 pts had objective responses, 2 of those had Notchmut (1 of which had ACC). ACC, a rare cancer that most commonly develops in the major salivary glands, but can also arise in minor salivary glands in the trachea, lacrimal gland, and other sites, is refractory to chemotherapy, with a high recurrence rate. Notchact mut are found in a subset of ACC pts (11%–22%), with particularly aggressive disease and poor prognosis. There is no proven active treatment for R/M ACC pts (PMID 27870570). Methods: ACCURACY (NCT03691207) is an open-label, single-arm, multicenter study of AL101 (4 mg IV QW) in pts with R/M ACC (bone-exclusive disease included) with known Notch1-4act mut. Pts with disease progression within 6 months of enrollment or newly diagnosed metastatic pts are allowed; pts who received > 3 prior systemic Tx are excluded. Primary endpoint: ORR by RECIST v1.1 (or modified MDA bone criteria), by independent review committee (IRC). Secondary endpoints: ORR by investigator review (IR), duration of response by IRC and IR, PFS by IRC, OS, and safety. Per the Simon optimal design, 12 pts are enrolled in stage 1; if ≥2 pts respond, 24 additional pts are enrolled in stage 2. If ≥4 pts in stage 2 respond, the trial is deemed positive. This design yields 5% type I error rate and 80% power, if ORR is 25%. 4 of planned 36 pts have been enrolled as of 2/12/19. Clinical trial information: NCT03691207.

Membrane dynamics of Notch-bound γ-secretase produces two distinct Notch conformations

Cleavage of Notch by the major intramembrane aspartyl protease complex γ-secretase is a central event in cell regulation and is also important to Alzheimer’s disease, with more than 200 mutations in the catalytic subunit of γ-secretase (PS1) causing severe early-onset forms of the disease. Recently, cryogenic electron microscopy (cryo-EM) has revealed the electron density of the protein-Notch complex in frozen solution, indicating major changes upon substrate binding and a possible helix unwinding to expose peptide bonds. In order understand the all-atom dynamics that cause this process, and to test the Notch binding in a membrane protein rather than solution, we developed an all-atom model of mature wild-type γ-secretase bound to Notch in a complete membrane-water system and studied the system using three independent 500-nanosecond molecular dynamics simulations. Our ensembles are in essential agreement with known cryo-EM data. As in previous simulations we find unusual β-strand transitions in exposed parts of PS1. We also observe the atomic helix motions that cause loss of helicity in bound Notch by direct comparison to corresponding 500 ns simulations of free Notch, in particular five residues to the N-terminal site of the primary cleavage site. Most importantly, we identify three conformation states, with two of them differing in the Notch-bound catalytic site. These dynamics produce a ping-pong relationship of positioning the S3 cleavage sites of Notch relative to the aspartates. These conformation states are not visible in the cryo-EM data; probably the density is an average snapshot of the two states. Our identified conformation states rationalize how Notch cleavage can be imprecise and yield multiple products. Our identified conformation states may aid efforts to develop conformation-selective drugs that target C99 and Notch cleavage differently.Statement of SignificanceThe atomic dynamics underlying cleavage of Notch by γ-secretase in the membrane is of major biological importance. Electron microscopy has revealed the protein-Notch complex in frozen solution, showing major changes upon substrate binding and helix unwinding to expose peptide bonds, but does not explain why substrate cleavage is imprecise and produces several products. Our model of wild-type γ-secretase bound to Notch in a complete membrane-water system equilibrated by 3 × 500 nanoseconds of molecular dynamics strongly complements the electron microscopy data: We identify the specific loop and helix motions that cause the β-strand transitions in PS1 and the loss of helicity in specific residues of bound Notch. We identify different conformations of Notch, which importantly affect the S3 cleavage site; the open state may cause the imprecise cleavage with earlier release of products. Our identified states can aid development of conformation-selective drugs that target C99 and Notch cleavage differently.

Drosophila IMP regulates Kuzbanian to control the timing of Notch signalling in the follicle cells

The timing of Drosophila egg chamber development is controlled by a germline Delta signal that activates Notch in the follicle cells to induce them to cease proliferation and differentiate. Here we report that follicle cells lacking the RNA-binding protein IMP go through one extra division due to a delay in the Delta-dependent S2 cleavage of Notch. The timing of Notch activation has previously been shown to be controlled by cis-inhibition by Delta in the follicle cells, which is relieved when the miRNA pathway represses Delta expression. imp mutants are epistatic to Delta mutants and give an additive phenotype with belle and dicer mutants, indicating that IMP functions independently of both cis-inhibition and the miRNA pathway. We find that the imp phenotype is rescued by over-expression of Kuzbanian, the metalloprotease that mediates the Notch S2 cleavage. Furthermore, Kuzbanian is not enriched at the apical membrane in imp mutants, accumulating instead in late endosomes. Thus, IMP regulates Notch signalling by controlling the localisation of Kuzbanian to the apical domain, where Notch cleavage occurs, revealing a novel regulatory step in the Notch pathway.SummaryIMP regulates Notch signalling in follicle cells by controlling Kuzbanian localisation to the apical domain, where Notch cleavage occurs, revealing a novel regulatory step in the Notch pathway.