Roeck, Kaely

Abstract BACKGROUND Intraductal papillary mucinous neoplasm (IPMN) is one of the two most common precursor lesions leading to the development of pancreatic ductal adenocarcinoma (PDA). IPMNs comprise a heterogeneous group of tumors with a wide range of grades and histotypes, and the emergence of single-cell RNA sequencing (RNA-seq) and multiplex digital spatial profiling have characterized unique cell populations, including dysplastic epithelial and immune cells, within the heterogeneous tumor microenvironment that carry signature gene expressions, which could be used as markers for disease progression. However, it is not clear on the heterogeneity of the epithelial and immune response of the evolution during progression of IPMN to PDA. OBJECTIVE Combining nuclear GSK-3β with oncogenic KRas mice (referred to as KNGC – KRas, nuclear GSK-3β, Cre) resulted in the development of IPMN. We have found that 4-week-old KNGC mice show progressive desmoplasia and ductal lesion development with increased M2-like macrophage infiltration. Interestingly, CX3CL1 (fractalkine), a chemokine involved in the recruitment of CX3CR1-expressing monocytes and M2-like macrophage conversion in various disease models is expressed by IPMN progenitor ductal cells. We aim to investigate expression and contribution of CX3CL1-CX3CL1 axis in the development and progression of IPMN. METHODS We utilized a newly generated CX3CL1-mCherry and well-established CX3CR1-GFP knockin/knockout mouse for crossbreeding with KNGC and KGC (KRas, GNas, Cre) for lineage tracing and function analysis by immunofluorescent staining, flow cytometry and CyTOF. RESULTS Both KNGC- and KGC-3LmCherry/+ mice harbor a large population of CX3CL1-mCherry/EPCAM+ epithelial cells as compared to 3LmCherry/+ and wildtype (WT) control mice. In KNGC and KGC mice, we show that there was a unique population of macrophages (CD45+ CD11b+ Ly6G- F4/80+ Ly6c- ) with significantly higher expression of CX3CR1 (CX3CR1Hi) that was not seen in WT mice. Significantly, Knockout of CX3CR1 in KNGC-3RGFP/GFP animals had a paucity of M2-like macrophages, reduced T regulatory cells (Tregs), substantially reduced desmoplasia and impaired development of IPMN. CONCLUSIONS The study provides important knowledge regarding the role of CX3CL1-CX3CR1 axis in recruitment and generation of M2-like macrophages, and their impact on the initiation of the immune/stroma microenvironment facilitating IPMN development. Further investigations using these models have the potential to identify new biomarkers for risk stratification and therapeutic intervention. Citation Format: Li Ding, Kaely Roeck, Easton Maeder, Cheng Zhang, Taylor Weiskittel, Esther Rodman, Mark Maynes, Bibo Zhu, Aaron Johnson, Jie Sun, Li Hu, Daniel Billadeau. Uncoupling CX3CL1-CX3CR1 Axis in IPMN Development Through Transgenic Mouse Models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A064.

Abstract Glycogen synthase kinase-3 (GSK-3) is overexpressed and accumulates in the nucleus in several human malignancies including pancreatic cancer. We recently demonstrated that GSK-3 inhibition sensitizes pancreatic cancer cells to chemotherapy by abrogating the TopBP1/ATR-mediated DNA damage response. Specifically, GSK-3 kinase activity is required to stabilize TopBP1 protein. Herein, we investigated the mechanism by which GSK-3 phosphorylation of TopBP1 prevents its degradation. Interestingly, GSK-3 inhibition not only decreased the total levels of TopBP1, but also reduced the ability of TopBP1 to bind chromatin following gemcitabine treatment. Mutation of a putative GSK-3 phosphorylation site in TopBP1 (S860A) resulted in decreased TopBP1 stability and chromatin recruitment, whereas a phosphomimetic mutant (S860E) had the opposite effect. Furthermore, we found enhanced TopBP1 ubiquitination after GSK-3 inhibition as well as an increased interaction between TopBP1 and hHYD, an ubiquitin ligase that has been shown to ubiquitylate TopBP1 resulting in its degradation. Significantly, the S860A mutation showed a stronger interaction with hHYD compared to WT or the S860E mutant of TopBP1. In conclusion, our study identifies GSK-3 phosphorylation of TopBP1 as a mechanism to prevent the interaction with and ultimate ubiquitylation-mediated degradation by hHYD. Citation Format: Li Ding, Kaely R. Roeck, Olga Alekhina, Daniel M. Schmitt, Daniel D. Billadeau. Glycogen synthase kinase-3 inhibition impairs DNA damage response through ubiquitin ligase hHYD-mediated TopBP1 degradation [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 4138.