Proline-Rich Tyrosine Kinase-2 (PYK2): A New Target for the Treatment of Hematopoietic Neoplasms with TEL-FGFR3 Fusion or FGFR3 Active Mutation.

Abstract

Constitutive activation of Fibroblast Growth Factor 3 (FGFR3) tyrosine kinase have been identified in various human cancers and have been reported to play an important role in some hematopoietic neoplasms. We have previously reported that TEL-FGFR3 in a patient with peripheral T-cell Lymphoma and AML conferred IL-3 independency to Ba/F3 cells and activates PLCγ, PK3K, STAT3, STAT5, MAPK through its constitutive tyrosine kinase activity in TEL-FGFR3 transfected Ba/F3 cells (TF-V5). In KMS-11, human multiple myeloma cell line which expresses constitutively active mutant FGFR3, activations of PI3K and STAT3 pathways have been reported. However, little is known about how FGFR3 tyrosine kinase (TK) activates these downstream molecules. Here, we show that PYK2, a member of focal adhesion kinases, plays a pivotal role for the activation of PI3K, STAT3 and STAT5 in FGFR3 oncogenic pathways, and is a candidate for therapeutic target. PP1/PP2, a kinase inhibitor of SRC and PYK2, inhibited the cell growth of TF-V5 and KMS-11 cells in a dose-dependent manner (IC50=15μM, 25μM respectively), not affecting the cell growth of IL-3 dependent Ba/F3 cells. Another specific SRC inhibitor did not affect the cell growth of TF-V5 and KMS-11 cells. TEL-FGFR3 transfection to Ba/F3 cells led to the overexpression of PYK2 but not FAK. Expression and phosphorylation of PYK2 were identified in KMS-11 cells. Immunoprecipitation analysis using FGFR3 TK inhibitor SU5402 showed that the activation of PYK2 which was recruited to FGFR3 was dependent on the kinase activity of FGFR3. The cell growth of TF-V5 was completely inhibited at the concentration of PP1/PP2(30μM), which inhibited auto-phosphorylation of PYK2. PP1/PP2 suppressed the activation of PI3K-ATK pathway and decreased expression of C-MYC, inducing G1-arrest of TF-V5. PP1/PP2 induced intrinsic apoptosis of TF-V5 and did not affect activation of BAX but decrease expression of BCL-2 and BCL-XL through inactivation of STAT3 and STAT5. PP1/PP2 also inhibited the activation of PI3K and STAT3 in KMS-11 cells, inducing G1-arrest and apoptosis. PP1/PP2 inhibited tyrosine kinase of PYK2 mesured by in vitro kinase assay (IC50=23μM, 13μM, respectively). Further PYK2 C-terminus Associated Protein (PAP) siRNA expression plasmid significantly decreased the proliferation of TF-V5 but not mock transfected Ba/F3 cells. Our data demonstrates that PYK2 is an attractive molecular target for FGFR3 associated hematopoietic neoplasm.