PTPN9 dephosphorylates FGFR2pY656/657 via interaction with ACAP1 and ameliorates pemigatinib effect in cholangiocarcinoma

Cholangiocarcinoma (CCA) is a highly aggressive and lethal cancer that originates from biliary epithelium. Systemic treatment options for CCA are currently limited, and the first targeted drug of CCA, pemigatinib, emerged till 2020 for CCA treatment by inhibiting FGFR2 phosphorylation. However, the regulatory mechanism of FGFR2 phosphorylation is not fully elucidated. Here we screened the FGFR2-interacting proteins and showed that protein tyrosine phosphatase N9 (PTPN9) interacts with FGFR2 and negatively regulates FGFR2 pY656/657 . Using phosphatase activity assays and modeling the FGFR2-PTPN9 complex structure, we identified FGFR2 pY656/657 as a substrate of PTPN9, and found that sec. 14p domain of PTPN9 interacts with FGFR2 via ACAP1 mediation. Co-expression of PTPN9 and ACAP1 indicates a favorable prognosis for CCA. Additionally, we identified key amino acids and motifs involved in sec. 14p-APCP1-FGFR2 interaction, including the “YRETRRKE” motif of sec. 14p, Y471 of PTPN9, as well as the PH and Arf-GAP domain of ACAP1. Moreover, we discovered that the FGFR2 I654V substitution can decrease PTPN9-FGFR2 interaction and thereby reduce the effectiveness of pemigatinib treatment. Using a series of in vitro and in vivo experiments including PDX, we showed that PTPN9 synergistically enhances pemigatinib effectiveness and suppresses CCA proliferation, migration, and invasion by inhibiting FGFR2 pY656/657 . In conclusion, our study identifies PTPN9 as a negative regulator of FGFR2 phosphorylation and a synergistic factor for pemigatinib treatment. The molecular mechanism, oncogenic function and clinical significance of PTPN9-ACAP1-FGFR2 complex are revealed, providing more evidence for CCA precision treatment.