Abstract 4387: Interplay of Notch1 and Notch3 signaling regulates pancreatic neuroendocrine tumor proliferation

Abstract Background: The 5-year survival rate for patients with advanced pancreatic neuroendocrine tumors (pNETs) is less than 30%. Notch signaling is a transmembrane receptor pathway with four receptor isoforms and five different ligands. Binding between a receptor and ligand triggers pathway activation and is linked to cellular differentiation, cell fate, proliferation, and viability. Both Notch1 and Notch3 signaling have been shown to be dysregulated in pNETs. Notch3 activation has been shown to repress Notch1 activity in other cancers. We hypothesize that activation of Notch3 can reduce Notch1-mediated proliferation in pNETs. Methods: Genetic deletion of Notch1 (N1-KO) in a pNET cell line (BON) was achieved using CRISPR/Cas9 to create a compound heterozygous knockout at exon 3. An inducible Notch1 overexpression cell line was generated by stably transfecting BON with a plasmid construct containing the Notch1 active form (N1ICD) under a Tet-On regulator. A constitutively active Notch3 cell line was generated by lentiviral transduction of BON with a plasmid overexpressing the Notch3 active form (N3ICD). Real-time quantitative PCR was used to measure mRNA transcript levels, and western blot was used to measure protein expression. Two-dimensional (2D) viability was measured by MTT assay, and three-dimensional (3D) proliferation was measured by spheroid size. Results: Genetic knockout of Notch1 (N1-KO) reduced the expression of Notch3 at both mRNA and protein levels. N1-KO had reduced proliferation compared to (WT) BON. Increased cell density (ligand availability) or treatment with valproic acid (known Notch1 inducer) led to increased N3ICD in WT BON but not in N1-KO. Overexpression of N1ICD was associated with increased expression of a known downstream target Hes1, as well as Notch3. In contrast, forced overexpression of N3ICD led to increased Notch1 expression, but not Hes1 when compared to a BON cell line transfected with an empty vector. Functionally, N1-KO and N3ICD overexpression cells both had reduced proliferation in both 2D and 3D cell culture. Conclusions: The Notch1 and Notch3 receptors produce signaling cascades that regulate growth in pNET cells through distinct mechanisms. Reduction of Notch1 signaling suppresses growth, while the same effect can be achieved with Notch3 overexpression. Thus, specifically upregulating Notch3 may be a strategy to block Notch1-mediated proliferation in pNET cells. Citation Format: Rachael Guenter, Weisheng Chen, Yuvasri Golivi, Mario Robledo, Cole Adams, Renata Jaskula-Sztul, Herbert Chen, J. Bart Rose. Interplay of Notch1 and Notch3 signaling regulates pancreatic neuroendocrine tumor proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4387.