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The Notch signaling pathway, particularly endothelial Notch signaling, plays a key role in tumor formation and angiogenesis. Inhibiting Notch1 or its ligand, Delta-like ligand 4 (Dll4), can induce hypersprouting of the tumor vasculature and thus can reduce tumor perfusion and progression. However, due to the expression of Notch1 in multiple healthy cell types, pharmacologic blockade poses the challenge of inducing tissue hypoxia, vascular neoplasms, and toxicity in non-target tissues. Unlike Notch1, Notch4 expression is largely restricted to endothelium and is upregulated in models of triple negative breast cancer (TNBC). Loss of Notch4 in mice results in delayed developmental angiogenesis and reduced mammary tumor angiogenesis and perfusion. Therefore, Notch4 is an attractive potential pharmacologic target to interfere with tumorigenesis and minimize nonspecific toxicity. We determined that Notch4 is expressed primarily in tumor endothelium in both human and mouse mammary tumors, including murine syngeneic Py8119 tumors, as well as a selection of other human and syngeneic mouse tumor types such as B16F10 melanoma tumors. We explored the effects of Notch4 inhibition on the progression of murine mammary epithelial carcinoma using newly developed anti-Notch4 antibodies, 6-3-A6 and its humanized derivative E7011. Administration of E7011 or 6-3-A6 to orthotopically implanted Py8119 murine breast carcinoma and B16F10 melanoma significantly reduced tumor size in comparison to IgG-treated controls. We performed single cell RNA sequencing of Py8119 tumors treated with E7011 and found that although Notch4 transcripts were found specifically in the endothelium, the most notable effect of E7011 was a dramatic increase in the number of macrophages and cancer-associated fibroblasts, suggesting a non-cell autonomous effect. To examine the potential role of these immune cell population changes in facilitating E7011’s anti-tumor activity, we investigated treatment of Py8119 murine mammary carcinomas in the absence of macrophages. Administration of clodronate liposomes to eliminate macrophages blocked the anti-tumor effects of E7011 treatment. Taken together, our data suggests that tumor endothelial Notch4 promotes TNBC growth by altering the immune landscape and increasing anti-tumor macrophage recruitment, and that blockade via the novel anti-Notch4 E7011 antibody has potential therapeutic efficacy. Citation Format: Jason W. Eng, L.A. Naiche, Bhairavi Swaminathan, Yu Kato, Krishna Thakkar, Katherine Ann Alexander, Rahul Vadakath, James H. Herts, Rajyasree Emmadi, Junji Matsui, Jan K. Kitajewski. Notch4 inhibition by a novel neutralizing antibody reduces tumor progression and increases macrophage recruitment within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB009.

Endothelial Notch signaling is critical for tumor angiogenesis and growth. Inhibitors of Notch1 or its ligand Dll4 cause tumor vessel hypersprouting but interfere with vessel perfusion causing reduced tumor growth. Unfortunately, normal tissue vessels respond to these inhibitors causing tissue hypoxia or vascular neoplasms. Mice null for Notch4, unlike Notch1 mutants, exhibit reduced developmental and tumor angiogenesis. Notch4 expression is increased in the endothelium of some tumor types. These results suggest that specifically targeting Notch4 could reduce tumor angiogenesis with fewer effects on normal tissue and represents an unexplored therapeutic opportunity. We use a newly developed anti-Notch4 antibody, E7011, and show that in endothelial cells, inhibition of Notch4 signaling suppresses expression of a subset of canonical Notch target genes. Treatment with E7011 also suppresses expression of a unique set of genes not regulated by canonical Dll4-Notch1 signaling, which appear to be distinct Notch4 targets. We administered E7011 to neonatal mice to examine the effects on developmental retinal angiogenesis. A single dose of E7011 significantly reduced retinal vascular outgrowth, demonstrating that E7011 has anti-angiogenic activity in vivo. E7011 treatment did not alter capillary density within the vascular plexus or endothelial tip cell number, both of which are typically increased by loss of Notch1 signaling. Because the E7011 phenotype was markedly distinct from the Notch1 loss of function, we combined E7011 treatment with endothelial-specific loss of Notch1 (Notch1ECKO) to compare Notch1 to Notch4 function. E7011-treated Notch1ECKO retina showed a compound phenotype, exhibiting both the loss of vascular outgrowth characteristic of E7011/Notch4 and the hypersprouting characteristic of Notch1 loss of function. These results suggest that Notch1 and Notch4 have distinct roles in developmental angiogenesis, with Notch4 being a pro-angiogenic signaling pathway. We evaluated Notch4 expression in the breast cancer tumor microenvironment to determine if Notch4 functions in tumor vessels. Human triple negative breast cancer (TNBC) shows a range of expression of Notch4 in both tumor cells and tumor endothelium. The anti-angiogenic effects that we observe in the retina lead us to hypothesize that E7011 will show anti-tumor activity against tumors that induce Notch4 expression in neighboring endothelium, and that targeting endothelial Notch4 is a novel anti-angiogenic approach. Citation Format: L. A. Naiche, Bhairavi Swaminathan, Yu Kato, Sarita Das, Jason W. Eng, Qanber Raza, Krishna Thakkar, James H. Herts, Rajyasree Emmadi, Junji Matsui, Jan K. Kitajewski. A novel Notch4 neutralizing antibody inhibits angiogenesis and tumor growth via a distinct mechanism from endothelial Notch1 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1488.

Introduction: Nitric oxide (NO) is known to inhibit vascular smooth muscle cell (VSMC) proliferation by modulating cell cycle proteins. The 26S proteasome tightly regulates the degradation of most cell cycle proteins. Therefore, we previously studied the relationship between NO and the 26S proteasome, and found that NO directly inhibits 26S proteasome catalytic activity. The purpose of this study was two-fold: to determine the effect of NO on 26S proteasome subunit expression, and to determine the intracellular localization of these subunits. Methods: Protein expression was examined using western blot analysis conducted on rat aortic VSMC exposed to varying concentrations of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 125–1000 μM) for 24 hours. Intracellular localization was conducted with immunofluorescent staining of VSMC ± exposure to SNAP (500 μM). VSMC were also fractionated into their nuclear and cytoplasmic components and western blot analysis was conducted to confirm localization. Results: Of the 26S proteasome subunits examined, the α5, α6, β1, and inducible β1 subunits demonstrated increased protein expression with increasing concentrations of NO. However, β2, inducible β2, β5, and inducible β5 protein expression did not change with NO exposure. Immunofluorescent staining of VSMC for these subunits confirmed these results. Additionally, each of these subunits showed a distinct pattern of staining: the α5 subunit was mostly perinuclear, the α6 subunit appeared mostly nuclear, and the β1 and inducible β1 subunits were mostly cytoplasmic. Western blot analysis following nuclear and cytoplasmic separation confirmed that α5, β1, and inducible β1 subunits were mostly in the cytoplasmic fraction. Interestingly, while the α6 subunit localizes to the nucleus by immunofluorescent staining, western blotting showed it to be mostly in the cytoplasmic fraction, indicating that its cellular distribution is perinuclear. Conclusion: Our data indicate that the 26S proteasome subunits have distinct patterns of intracellular localization, and are differentially regulated by NO. Understanding the relationship between NO and the 26S proteasome provides insight into the mechanism by which NO inhibits VSMC proliferation.