<div>Abstract<p>Endothelial Notch signaling is critical for tumor angiogenesis. Notch1 blockade can interfere with tumor vessel function but causes tissue hypoxia and gastrointestinal toxicity. Notch4 is primarily expressed in endothelial cells, where it may promote angiogenesis; however, effective therapeutic targeting of Notch4 has not been successful. We developed highly specific Notch4-blocking antibodies, 6-3-A6 and humanized E7011, allowing therapeutic targeting of Notch4 to be assessed in tumor models. Notch4 was expressed in tumor endothelial cells in multiple cancer models, and endothelial expression was associated with response to E7011/6-3-A6. Anti-Notch4 treatment significantly delayed tumor growth in mouse models of breast, skin, and lung cancers. Enhanced tumor inhibition occurred when anti-Notch4 treatment was used in combination with chemotherapeutics. Endothelial transcriptomic analysis of murine breast tumors treated with 6-3-A6 identified significant changes in pathways of vascular function but caused only modest change in canonical Notch signaling. Analysis of early and late treatment timepoints revealed significant differences in vessel area and perfusion in response to anti-Notch4 treatment. We conclude that targeting Notch4 improves tumor growth control through endothelial intrinsic mechanisms.</p>Significance:<p>A first-in-class anti-Notch4 agent, E7011, demonstrates strong antitumor effects in murine tumor models including breast carcinoma. Endothelial Notch4 blockade reduces perfusion and vessel area.</p></div>
<p>Treatment of human tumor xenografts with 6-3-A6 delays growth of tumors which express endothelial Notch4. <b>A,</b> IF staining of Notch4 (red) and CD31<sup>+</sup> tumor vessels (green) in human tumor xenografts. Arrowheads indicate Notch4-expressing vessels (Scale bar = 200 μm). <b>B</b> and <b>C,</b> Tumor growth curves of subcutaneously implanted tumors from the indicated cell lines. <b>B,</b> MDA-MB-231 xenografts treated intravenously with 6-3-A6 (<i>n</i> = 5). <b>C,</b> Calu-6 human NSCLC xenograft tumors treated intravenously with 6-3-A6. All treatments started 12 hours after tumor implantation. Statistical analysis performed with two-way ANOVA for tumor growth curves, plotted as average ± SEM; *, <i>P</i> < 0.05.</p>
<p>Notch4 is expressed on tumor ECs, and treatment alters the tumor vessel transcriptional profile. <b>A,</b> Representative flow plots of gating for CD31<sup>+</sup> cells in Py8119 and B16F10 tumors. <b>B,</b> Histograms comparing E7011 and human IgG binding to CD31<sup>+</sup> and CD31<sup>−</sup> cell populations from dissociated Py8119 and B16F10 tumors (<i>n</i> = 3). Left, E7011 samples were concatenated and merged into a single representative histogram. Right, Graph of concatenated MFI from Py8119 and B16F10 samples bound with E7011 or human IgG. <b>C,</b> Heatmaps of significantly upregulated and downregulated differentially expressed genes isolated from IP polysomes from tumor ECs treated with 6-3-A6 or control IgG (<i>n</i> = 3). Boxes indicate genes implicated in GPCR control of angiogenesis (red), endothelial nitric oxide regulation (blue), and inflammatory/immune response (green). <b>D,</b> Reactome pathway analysis of upregulated and downregulated pathways was performed using g:Profiler, and significance threshold by Benjamini–Hochberg FDR was set at 0.05. <b>A,</b> Fold cutoff of −log<sub>10</sub>(<i>P</i><sub>adj</sub>) > 2 was used to select the displayed pathways. Statistical analysis performed with the <i>t</i> test for perfusion studies, plotted as average ± SEM; *, <i>P</i> < 0.05; **, <i>P</i> < 0.005. Heatmap data presented as <i>Z</i>-scores, which integrate both fold change and significance.</p>
<p>A, Schematic of experimental design of Py8119 orthotopically implanted tumors in RiboTag<sup>EC</sup> mice. Female RiboTag<sup>EC</sup> mice with 75mg/kg of tamoxifen (20mg/mL) for 5 days by IP injection. Py8119 tumors were implanted at 8 weeks of age and then treated once tumors reached 50mm3 with twice weekly IV control IgG or 6-3-A6 for a total of 10 days (n=3). B, Growth curve of RiboTagEC treated with 25mg/kg of IV control IgG or 6-3-A6 twice weekly for 10 days (n=3). C, Heatmap of endothelial expressed genes segregated by total mRNA input and ribosomal IP mRNA from control IgG treated and 6-3-A6 treated Py8119 tumors. D, PCA plots of the ribosomal IP fraction from control IgG and 6-3-A6 treated tumors. Two samples from the control IgG and 6-3-A6 groups were identified as outliers and removed from analysis. E, Heatmap of downstream Notch regulated genes from endothelial IP of the control IgG or 6-3-A6 treated tumors and quantitative rt-PCR of the canonical Notch target gene <i>Hes1</i> and <i>Rnd1</i> amplified from 6-3-A6 treated and control RiboTag IP fractions (n=4). F, Heatmaps of genes associated with significantly enriched or downregulated signaling pathways involved in inflammation and extracellular matrix interactions. All heatmap data presented as Z-scores. Statistics by t-test for qPCR studies</p>
L.A. Naiche1,3, Stephanie R. Villa1,3 and Jan K. Kitajewski1,2 1Department of Physiology and Biophysics, University of Illinois Chicago, Chicago, Illinois 60612, USA 2University of Illinois Cancer Center, Chicago, Illinois 60612, USA Correspondence: kitaj{at}uic.edu ↵3 These authors contributed equally to this work.
Abstract Notch signaling is activated by ligands Delta-like 4 (Dll4) and Jagged1 in the tumor microenvironment to promote tumor angiogenesis and perfusion. Notch activation is associated with poor outcomes in several cancers, particularly triple negative breast cancer (TNBC), and affects both tumor angiogenesis and metastasis. The development of therapeutics targeting angiogenesis, such as the Notch pathway, has again attracted attention. Previous approaches to globally inhibit the Notch pathway or block Dll4/Notch1 activation, such as γ-secretase inhibitors (GSI), have raised safety concerns due to gastrointestinal toxicity due to accumulation of secretory goblet cells in the intestine. Similarly, anti-Dll4 therapy resulted in pathological changes in the liver as well as severe vascular neoplasms when evaluated using preclinical animal models. Development of new approaches for targeting the Notch pathway remains a critical clinical problem currently unaddressed. Our lab has previously developed ligand-specific inhibitors of Notch signaling, called Notch decoys, which are comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain. These Notch decoys bind ligand non-productively and interfere with ligand function. Jagged-specific Notch1 decoys inhibit angiogenesis in vitro and significantly impair tumor growth, tumor angiogenesis, and perfusion without apparent toxicity in mouse models of TNBC. However, this previous work utilized Notch decoys produced via viral expression vectors, which preclude dosage control and limit clinical applicability. We have developed a new generation of Notch “mini” decoys that contain fewer EGF-like repeats. These mini decoys show improved secretion characteristics and can be purified as active proteins in clinically relevant quantities. Using a variety of binding assays, we observed that these Notch mini decoys demonstrate strong but distinct binding to Notch ligands Dll4 and Jagged1 and block Notch signaling when evaluated in cultured cells. Our newly generated Notch decoys can now be sufficiently purified for use in a dose-dependent manner to test the therapeutic role of Notch inhibition on tumor angiogenesis using preclinical animal models. Citation Format: Timothy Sargis, Seock-Won Youn, Hyun Lee, L.A. Naiche, Jan Kitajewski. Notch decoys as potential anti-angiogenic biotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3207.
Abstract Notch signaling guides vascular development and function by regulating diverse endothelial cell behaviors, including migration, proliferation, vascular density, endothelial junctions, and polarization in response to flow. Notch proteins form transcriptional activation complexes that regulate endothelial gene expression, but few of the downstream effectors that enable these phenotypic changes have been characterized in endothelial cells, limiting our understanding of vascular Notch activities. Using an unbiased screen of translated mRNA rapidly regulated by Notch signaling, we identified novel in vivo targets of Notch signaling in neonatal mouse brain endothelium, including UNC5B , a member of the netrin family of angiogenic-regulatory receptors. Endothelial Notch signaling rapidly upregulates UNC5B in multiple endothelial cell types. Loss or gain of UNC5B recapitulated specific Notch-regulated phenotypes. UNC5B expression inhibited endothelial migration and proliferation and was required for stabilization of endothelial junctions in response to shear stress. Loss of UNC5B partially or wholly blocked the ability of Notch activation to regulate these endothelial cell behaviors. In the developing mouse retina, endothelial-specific loss of UNC5B led to excessive vascularization, including increased vascular outgrowth, density, and branchpoint count. These data indicate that Notch signaling upregulates UNC5B as an effector protein to control specific endothelial cell behaviors and inhibit angiogenic growth.
Abstract To control sprouting angiogenesis, endothelial Notch signaling suppresses tip cell formation, migration, and proliferation while promoting barrier formation. Each of these responses may be regulated by distinct Notch-regulated effectors. Notch activity is highly dynamic in sprouting endothelial cells, while constitutive Notch signaling drives homeostatic endothelial polarization, indicating the need for both rapid and constitutive Notch targets. In contrast to previous screens that focus on genes regulated by constitutively active Notch, we characterized the dynamic response to Notch. We examined transcriptional changes from 1.5 to 6 h after Notch signal activation via ligand-specific or EGTA induction in cultured primary human endothelial cells and neonatal mouse brain. In each combination of endothelial type and Notch manipulation, transcriptomic analysis identified distinct but overlapping sets of rapidly regulated genes and revealed many novel Notch target genes. Among the novel Notch-regulated signaling pathways identified were effectors in GPCR signaling, notably, the constitutively active GTPase RND1 . In endothelial cells, RND1 was shown to be a novel direct Notch transcriptional target and required for Notch control of sprouting angiogenesis, endothelial migration, and Ras activity. We conclude that RND1 is directly regulated by endothelial Notch signaling in a rapid fashion in order to suppress endothelial migration.
<p>Development and validation of 6-3-A6 and E7011 as Notch4 inhibitors. <b>A,</b> Schematic of the immunizing epitope used to generate clone 6-3-A6. EGF, EGF repeats; Igk, immunoglobulin κ chain; SEAP, secreted embryonic alkaline phosphatase. <b>B,</b> Binding assay of the fully humanized E7011 and 6-3-A6 fused to human Fc for immobilization. Affinity to recombinant human Notch4 was calculated with surface plasmon resonance. <b>C,</b> Affinity of 6-3-A6 and E7011 to Raji cell lines generated to overexpress full-length human Notch1, Notch2, Notch3, and Notch4 (hNotch1 to 4, respectively). Binding was determined by MFI with flow cytometry analysis (<i>n</i> = 3). <b>D,</b> Luciferase reporter assay using a Notch4-ECD/Notch1-ICD-Gal4 fusion protein expressed in bEnd.3-immortalized ECs. Luminescence was measured with increasing concentrations of added E7011 or 6-3-A6. Statistical analysis performed with one-way ANOVA with the Dunnett multiple comparison test, plotted as average ± SEM; **, <i>P</i> < 0.01; ****, <i>P</i> < 0.0001.</p>
