A106-04 Enhanced Fibroblast Growth Factor Receptor 2 Signaling Disrupts Tracheal Epithelial Cell Differentiation in Pediatric Patients

Rationale Enhanced Fibroblast Growth Factor Receptor 2 (FGFR2) signaling is strongly associated with severe developmental airway abnormalities, notably Tracheal Cartilaginous Sleeve (TCS), which carries high morbidity and mortality due to airway obstruction in children with syndromic craniosynostoses. These patients have abnormal mucociliary clearance, recurrent infections, and need for tracheostomy for airway patency. Yet, the mechanism by which these FGFR2 mutations alter airway development and function remains a knowledge gap. We hypothesize that airway epithelial cell (AEC)-derived FGFR2 signaling is the primary driver of this pathology; mechanistic understandings of AEC dysfunction in these patients will inform development of targeted future therapies. Methods Primary AECs were prospectively collected via tracheal brushings from well-characterized and genotyped pediatric patients: those with confirmed FGFR2 enhanced signaling (FGFR2-ES) mutations (n = 3) and non-syndromic comparator patients undergoing bronchoscopy (n = 3). Cells were expanded and differentiated at air-liquid interface (ALI) for 21 days to form organotypic AEC cultures. We performed H&E and immunofluorescence on formalin-fixed paraffin-embedded ALI cultures to assess epithelial layer thickness and cell type composition. Ciliated cell area percentage was determined by quantitative confocal microscopy of whole-mount immunofluorescence. Bulk RNA sequencing (RNA-seq) was conducted on proliferating and fully differentiated ALI cultures (n = 2 FGFR-ES and comparators). Prism was used to evaluate for statistical differences with nonparametric tests. Results Compared to comparator AECs, differentiated FGFR2-ES AEC cultures showed marked alterations in epithelial structure. Histological examination demonstrated a thinner epithelial cell layer in FGFR2-ES patient samples. Immunofluorescence staining for cell-specific markers demonstrated a shift in cell-type composition, with a notable increase in CC10+ club cells and MUC5A+ goblet cells in FGFR2-ES AECs. Ki67 staining showed reduced basal cell proliferation in the FGFR2-ES AECs. Quantitative microscopy revealed decreased ciliated cell proportions in FGFR2-ES cultures (p = 0.1). RNA-seq analysis confirmed differences in key developmental and signaling pathways, consistent with a disruption in the normal differentiation program. Proliferative AECs from patients with FGFR2-ES mutations expressed 20.2-fold less TBL1Y and 1.8-fold less CTBP1 than comparator AECs, suggesting dysregulation of Wnt and Notch differentiation pathways. Conclusions Activating mutations in FGFR2 lead to altered airway epithelial cell proliferation and differentiation, uncovering a potential mechanism for increased mucous production and impaired mucociliary clearance in these patients. This cell-autonomous epithelial dysfunction may also represent a critical pathogenic event in FGFR2-related airway maldevelopment. These findings establish a crucial mechanistic link between genotype and phenotype in patients with activating FGFR2 mutations and thereby provide a foundation for discovery of targeted airway therapies. This abstract is funded by: Seattle Children’s Research Institute