Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies

Abstract

During embryonic development, cell fate decisions are orchestrated through spatiotemporal modulation of extracellular cues. The precise molecular mechanisms by which cells decode these signals to regulate lineage commitment remain an outstanding question in developmental biology. Growth factors and cytokines initiate intracellular signaling cascades by engaging the extracellular domains of receptor tyrosine kinases (RTKs), facilitating receptor oligomerization and transphosphorylation of intracellular kinase domains. The Fibroblast Growth Factor (FGF) signaling axis, in particular, is a key determinant of cell fate and survival, yet the isoform-specific functions of alternatively spliced FGF receptor variants during development remain poorly understood. To systematically interrogate the role of FGFR valency and geometry in signaling dynamics, we engineered synthetic cyclic homo-oligomers derived from repeat protein building blocks. These scaffolds were functionalized with a de novo designed binding module specific for the c-splice variant of FGFR1/2, enabling controlled activation. These synthetic FGFR1/2c signaling ligands exhibited potent valency- and geometry- dependent Ca2+ release and MAPK pathway activation. We also showed that these synthetic agonists have a capacity to drive endothelial cell differentiation through an FGF-mimetic trajectory. By leveraging these tools, we delineated isoform-specific roles of FGFR variants in early vascular development. Selective activation of the c-isoform promoted endothelial cell specification, with a marked bias toward arterial endothelial cell fate, whereas antagonists selective for the b-isoform preferentially directed differentiation toward perivascular cell lineages. Endothelial cells derived from human iPSCs using the c-isoform agonists were functionally mature, integrating seamlessly into vascular networks in vivo. These findings demonstrate the capability of engineered FGFR1/2c agonists to dissect FGFR variant functions in development, offering a tool to untangle signaling complexities during critical developmental transitions. Furthermore, leveraging computationally designed PRC2 inhibitors fused to dCas9, we demonstrate the targeted upregulation of p16 in diffuse midline gliomas, resulting in a significant reduction in tumor cell viability. These findings underscore the therapeutic potential of epigenetic reprogramming in cancer.