Abstract 844: Investigating the ability of <i>foxo1</i> to maintain stem cells.

Abstract Background: Our group discovered that FOXO transcription factors drive stem gene expression in cancer to promote aggressiveness. Work by our group and others demonstrated that FOXO factors universally maintain stem cells including in cancer, embryonic, hematogenic and neuronal contents. Our current efforts are delineating the molecular underpinnings by which FOXO factors halt differentiation to maintain stem cells. In this project, we are employing myoblasts as a valuable model to examine how Foxo1 maintains stem cells. Foxo1-regulated genes in myoblasts closely parallel its targets in glioblastoma and basal breast cancer stem cells, including targets such as Leukemia Inhibitory Factor, Lif, encoding a cytokine that prevents differentiation. Understanding how Foxo1 maintains stem cells is crucial, as it holds the potential to uncover new therapeutic options that could target the fundamental biological processes involved in cancer progression and chemotherapeutic resistance. Methods: RNAi was performed on C2C12 myoblasts cells to create Foxo1 knockdown and control lines, followed by assessment of target genes involved in differentiation, proliferation, and muscle fiber types using qRT-PCR. Fluorescent microscopy was utilized to investigate the structural differences in actin filaments and nuclei between Foxo1 knockout cells and the control group. Our initial work, including investigating candidate genes using qRT-PCR. We are currently taking genomics approaches to investigate the role of Foxo1 in myoblast/stem cell differentiation. Results: ​ ​In Foxo1 knockout cells, there was a notable decrease in Lif expression, suggesting differentiation.​ Downregulation of genes like Igfbp1, Socs1, and Socs2 indicates possible direct or indirect activation by Foxo1, with implications for cell growth and cytokine signaling. Additionally, downregulation of Pepck and the modulation of energy metabolism pathways suggest altered metabolic states. The downregulation of Stat1 and Wnt3 implies disruptions in pathways that promote proliferation to favor differentiation. This aligns with observations of increased nuclei and actin filament abundance in Foxo1 knockout cells, reflecting enhanced muscle differentiation and myotube formation. Conclusion: Foxo1’s involvement in regulating cellular dynamics through gene expression is complex and dynamic, impacting differentiation and proliferation, with notable changes in the cytoskeleton facilitating myoblast fusion and differentiation. Gaining insights into the relationship between Foxo1 and target genes will clarify potentially conserved roles that determine the differentiation status of stem and progenitor cells. We are anticipate that this model system will allow us to glean fundamental mechanisms that maintain stems cells to ultimately target cancer stem cells, which are known to trigger recurrence and chemotherapeutic resistance. Citation Format: Stella Aime Rios, Megan E. Keniry. Investigating the ability of foxo1 to maintain stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 844.