The oncogenic activation of receptor tyrosine kinases (RTK) promotes growth, survival and dissemination in pediatric tumors including glioma, ependymoma and medulloblastoma (MB). Direct targeting of either the RTK or of downstream kinases can effectively block tumor promoting pathway functions. However, emergence of resistance is common. We hypothesized that alternative interference strategies that target protein-protein interactions (PPIs) instead of enzymatic activities could overcome the emergence of resistance. We characterized the molecular interactions downstream of the FGFR that regulate relevant growth and invasion-promoting mechanisms in MB cells, to identify potentially druggable PPIs. We found that the FRS2 protein is an essential up-stream effector of FGFR signaling towards invasiveness. Using a proteomics approach, we furthermore identified the Striatin 3 protein as a novel oncogenic effector of the FGFR pathway downstream of FRS2, as it integrates antagonistic growth and invasion signals downstream of FGFR. Mechanistically, Striatin 3 interacts with the Ser/Thr kinase MAP4K4, couples it to the protein phosphatase 2A, and thereby inactivates growth repressing activities of MAP4K4. In parallel, Striatin 3 enables MAP4K4-mediated phosphorylation of PKC-theta and VASP, which combined are necessary to promote tissue invasion. To selectively repress pro-invasive FGFR functions, we identified and functionally validated small molecule ligands of FRS2, that prevent FRS2 activation and downstream signaling. We demonstrate efficacy of these compounds in inhibiting invasion and growth promoting activities in vitro and in vivo, and identified potential off-target activities of the ligand using a proteome-wide interaction analysis. We propose inhibition of FRS2 by a small molecular PTB domain ligand as a strategy to repress FGF signaling in FGFR-driven tumors. The development of this ligand, and the de novo design of functional analogs thereof bear promise for further pre-clinical evaluation of these structures as anti-growth promoting and anti-metastatic therapeutics applicable to FGFR-driven tumors.
Rational targeting of proteins involved in controlling cancer cell behavior with small bioactive compounds can accelerate anti-cancer drug discovery. We report the identification of a new small molecule compound inhibitor of the FGFR adaptor protein FRS2. Pharmacophore-based computational screening combined with functional, biophysical, and structural binding analyses, led to the identification of low-molecular weight ligands that interact with the PTB domain of FRS2. By assessing the compounds’ anti-invasion activity in human FGFR-driven cancer cell models, three chemically distinct bioactive molecules were shortlisted for further analysis. A lead compound was selected that specifically repressed FGFR-driven MAPK activation and matrix invasion and displayed on-target activity in cells. Proteome-wide off-target activity of the primary lead was determined and functional in vivo efficacy in an FGFR driven ovarian cancer model confirmed. We propose inhibition of FRS2 by a small molecular PTB domain ligand as a strategy to repress FGF signaling in FGFR-driven human cancers.
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