Enhanced Notch Signaling Skews Hematopoietic Stem Cell Differentiation in Fanconi Anemia Murine Models

Abstract 1191 Objective: Hematopoietic stem cells (HSCs) can either self-renew or differentiate into various types of cells of the blood lineage. Little is known about the signaling pathways that regulate this choice of self-renewal versus differentiation. We studied the effect of altered Notch signaling on HSC differentiation in mouse models of Fanconi anemia (FA), a genetic disorder associated with bone marrow failure and progression to leukemia and other cancers. Methods: The study used a Notch reporter mouse, in which Notch-driven GFP expression acts as a sensor for HSC differentiation. Long-term hematopoietic stem cell (LT-HSC) and multipotential progenitor (MPP) cell compartments, as well as GFP expression in different cell populations were detected by Flow Cytometry analysis using primary bone marrow cells from Notch-eGFP-WT, Notch-eGFP- Fanca −/− or Notch-eGFP- Fancc −/− mice. Cell Cycle analysis was performed to distinguish the difference of quiescent state in GFP-gated LSK cells from these Notch-eGFP reporter mice. Colony forming units (CFU) assay and bone marrow transplantation (BMT) were utilized to determine HSC self-renew capacity. Gene arrays for pathways involved in DNA repair, cell cycle control, anti-oxidant defense, inflammatory response and apoptotic signaling were employed to define the gene expression signatures of the MPP population. Results and conclusions: In mice expressing a transgenic Notch reporter, deletion of the Fanca or Fancc gene enhanced Notch signaling in MPPs, which was correlated with decreased phenotypic long-term HSCs and increased formation of MPP1 progenitors. Furthermore, we found a functional correlation between Notch signaling and self-renewal capacity in FA hematopoietic stem and progenitor cells (HSPCs). Significantly, we show that FA deficiency in MPPs deregulates a complex network of genes in the Notch and canonical NF-kB pathways. Specifically, enhanced Notch signaling in FA MPPs was associated with the unregulation of genes involved in inflammatory and stress responses (including Rela, Tnfrsf1b, Gadd45b, Sod2, Stat1, Irf1 and Xiap ), cell-cycle regulation (including Ccnd1, Cdc16, Cdkn1a, Gsk3b, Notch2 and Nr4a2 ), and transcription regulation (including Rela, Stat1, Hes1, Hey1, Hoxb4, Notch1 and Notch2 ). Consequently, TNF-a stimulation enhanced Notch signaling of FA LSK cells, leading to decreased HSC quiescence and compromised HSC self-renewal. Finally, genetic ablation of NF-kB reduced Notch signaling in FA MPPs to nearly wide-type level, and blocking either NF-kB or Notch signaling partially restored FA HSC quiescence and self-renewal capacity. Translational Applicability: The study identifies a functional interaction between the FA pathway and Notch signaling in HSC differentiation and establishes a role of FA proteins in the control of balance between renewal and lineage commitment, hence contributing to hematopoiesis. These findings indicate that the Notch signaling pathway may represent a novel and therapeutically accessible pathway in FA. Disclosures: No relevant conflicts of interest to declare.