Virginia Research Day 2021

NF-kB INDUCING KINASE CRITICAL FOR GASTROINTESTINAL HOMEOSTASIS: DYSREGULATION OF NONCANONICAL NF-kB PATHWAY IMPLICATED IN COLITIS-ASSOCIATED COLORECTAL CANCER Holly A. Morrison 1 , Audrey Rowe 1 , Katherine Baumgarner 2 , Kristin Eden 1 , Daniel Rothschild 1 , Eda Holl 3 , Jon Hagar 2 , Stephan Brown 2 , Irving C. Allen 1 1 Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA, 2 Edward Via College of Osteopathic Medicine, Spartanburg, SC, 3 Duke University, Durham, NC 6 8 Clinical Scores Nikfl/fl x Lys+ Nikfl/fl

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ABSTRACT Colitis-associated colorectal cancer (CAC) is characterized by inflammation-induced carcinogenesis in the colon and rectum. Here, dysregulation of inflammatory signaling cascades, such as NF-kB signaling, drives the initiation of neoplastic transformation by increasing rates of DNA damage through ROS/RNS associated DNA breaks and SNPs, upregulating the production of proinflammatory cytokines, altering gut microbiota, and hindering DNA repair mechanisms. These harmful effects ultimately culminate to cause otherwise normal cells to have increased susceptibility to mutagenesis. Our research focuses on the understudied noncanonical NF-kB signaling pathway and how impairment of this signaling results in the formation of CAC. NF-kB inducing kinase (NIK) is a pertinent enzyme in this signaling cascade and is required for further activation of the noncanonical NF-kB signaling pathway . We hypothesize that dysregulated noncanonical NF-kB signaling generates a pro- tumorigenic microenvironment that induces intestinal epithelial cells (IECs) to acquire oncogenic mutations that promote de-differentiation into a stem-like state . In our research, we observe that complete knockout of the noncanonical pathway (i.e. Nik-/-) in mice results in significant changes in large intestine phenotypes, including reduced stem cell marker expression, diminished regeneration/differentiation capacity upon inflammatory conditions, and increased susceptibility towards inflammation-induced carcinogenesis. Following this preliminary research, novel tissue-specific knockout mice were generated to elucidate the mechanisms relating to the observed phenotypes. Translating this work to clinical relevancy, human colonic biopsy samples from colorectal cancer patients show significant downregulation of the noncanonical NF-kB pathway. Together, this data suggests that the noncanonical NF-kB pathway is critical for maintaining homeostasis in the GI tract and withstanding the damaging effects associated with cancer pathogenesis.

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Figure 7. Loss of NIK in myeloid cells show no significant effect on tumorigenesis in the colon. Clinical scores (composite score of weight loss, fecal consistency, rectal bleeding, and overall behavior; see Methods) between Nikfl/fl x LysM-cre mice and Nikfl/fl littermates were only rarely significantly different and there was no significant difference in weight loss tracking (A, B). Nikfl/fl x LysM-cre mice mice had significantly increased post-DSS colonic length (**p = 0.0036) (C).

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Figure 4. Confirmation of Bioinformatics Data in Local Patient Populations. Noncanonical NF-κB signaling was assessed in colon biopsies from local patient populations. n=8 normal control subjects; n=6 colorectal cancer patients.**p<0.01; ***p<0.005 Colonic crypts of Nik Whole-body Knockout Mice Display Decreased Levels of Proliferation and Stemness

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Figure 1. NF- κ B Signaling. There are 2 arms in NF-kB signaling: canonical and noncanonical signaling.

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Intestinal Epithelial Cell Specific NIK Knockout Mice Display Increased Susceptibility to Colorectal Tumorigenesis Figure 5. Decreased Stem Cell Proliferation in the Base of Colonic Crypts of Nik -/- mice. Assessments revealed significantly decreased Ki67 staining (stem cell and proliferation marker), as well as reduced Lgr5 expression (stem cell marker). Crypts express significantly higher Krt20 expression (marker for differentiation). Nik -/- mice compared to wild type controls (confirmed with Ki67 staining) *p<0.05

Background & Hypothesis

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Background: • Colorectal cancer = 2 nd most deadly/most common cancer in U.S. • Colitis-associated Colorectal cancer = inflammation- induced cancer • IBD patients with medical history of chronic inflammation 2-3 more likely to develop CAC Objective: Determine the cell-type of origin for colitis-associated colorectal cancer Hypothesis: We hypothesize that dysregulated noncanonical NF-kB signaling generates a pro-tumorigenic microenvironment that induces intestinal epithelial cells (IECs) to de- differentiate into a stem-like state.

Acknowledgements § Allen Lab: Rebecca Brock, Alissa Hendricks, Margaret Nagai-Singer, Brie Trusiano, Juselyn Tupik , Hannah Ivester, Khan Imran, Endia Fletcher, Audrey Rowe § This work was supported by the NIH/NIDDK (DK092355), VCOM One Health Seed Grant, and a Virginia Tech CVM-IRC pilot grant. Future Directions • Indicate susceptible cell population for early detection of disease. • Assess IECs enhanced stemness in proliferation and apoptosis assays • Evaluate NIK protein interactions & implication on downstream signaling Conclusion: Revealed previously unstudied role of noncanonical NF-κB signaling in colonic stem cell niche and implications on CAC development using mouse models of inflammation-induced colitis and human colorectal cancer patients. Figure 8. Loss of NIK in intestinal epithelial cells result in increased formation of tumorous growths with increased gene expression related to stem cell functions. Results from a Mouse Stem Cell Superarray show Nik x Villin polyps to have largely increased expression of stem cell markers and decreased expression of differentiation markers (Krt15) compared to Nikfl/fl polyps. Evidence that polyps may form from IECs with enhanced stemness, as NV mice were more susceptible to AOM/DSS model and had increased polyp count. . Conclusion & Future Directions

Figure 2. Schematic for Inflammation- driven Tumorigenesis. Chronic inflammation sustained for periods longer than several months ultimately results in promoting proliferative and survival signaling representative of carcinogenesis

Mouse Model & Methods

Figure 6. Loss of NIK in the epithelial cell compartment results in increased tumorigenesis in the colon. Clinical scores and weight loss tracking between Nik fl/fl x Villin-cre mice and Nik fl/fl littermates were only rarely significantly different (A, B, asterisks) and there were no changes in post-DSS colonic length (C) Nik fl/fl x Villin-cre mice showed significantly increased polyp formation (D, G) and a slight trend towards increased polyp size (E) compared to Nik fl/fl mice. Histologically, these polyps were determined to represent adenocarcinomas (H). Statistics were performed using the student’s t test and significant was defined as p ≤ 0.05; n=5-7 mice

Figure 3. AOM/DSS Model for Colitis-Induced Carcinogenesis. Mice are injected IP with AOM and administered 2.5% DSS in their drinking water for 3 cycles. Mice are weighed daily to measure percent body weight loss and scored daily for key clinical symptoms, including altered stool consistency, bleeding, gait, dehydration, grooming, antisocial behavior, and rectal prolapse.

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