ZNF198-FGFR1 Transforms Ba/F3 Cells to Growth Factor Independence and Results in High Level Tyrosine Phosphorylation of STATS 1 and 5

Abstract

The ZNF198- FGFR1 fusion gene arises as a result of the t(8;13)(p11;q12) in the 8p11 myeloproliferative syndrome. To determine the transforming properties of this chimeric protein we transfected ZNF198-FGFR1 into the interleukin (IL)-3 dependent cell line Ba/F3. Growth factor independent subclones were obtained in which ZNF198-FGFR1, STAT1, and STAT5 were constitutively tyrosine phosphorylated, as determined by immunoprecipitation and Western blot analysis. To test the hypothesis that constitutive activation of ZNF198-FGFR1 tyrosine kinase activity is a result of self-association of the fusion protein, we in vitro transcribed and translated ZNF198-FGFR1 and a derivative construct, ZNF198- FGFR1deltaC-myc, in which the C-terminal FGFR1 epitope was replaced by a c-myc tag. As expected, an anti-FGFR1 antibody immunoprecipitated ZNF198-FGFR1 but not ZNF198-FGFRdeltaC-myc. However when both products were translated together, both were coimmunoprecipitated by anti-FGFR1 antisera. Similar results were obtained by using an anti-myc antibody and demonstrated a physical interaction between the two proteins. Analysis of COS-7 cells transfected with ZNF198-FGFR1 demonstrated that the fusion gene, in contrast to normal FGFR1, is located in the cytoplasm. We conclude that ZNF198-FGFR1 is a cytoplasmic protein that self-associates and has constitutive transformation activity. These data suggest that ZNF198-FGFR1 plays a primary role in the pathogenesis of the t(8;13) myeloproliferative syndrome and is the first report to implicate STAT proteins in FGFR1-mediated signaling.

Critical Role of STAT5 Activation in Transformation Mediated by ZNF198-FGFR1

“…Plasmids-The ZNF198-FGFR1 and BCR-FGFR1 constructs in pcDNA3.1 have been described previously (8,29). The coding sequence for human FGR1 was similarly subcloned into pcDNA3.1.…”

“…For each transfection, 4 ϫ 10 6 cells were mixed with 16 g of plasmid DNA and subjected to electroporation using a Bio-Rad gene pulser set at 250 V and 960 F. After transfection, cells were cultured for 48 h before selection with either G418 1 mg/ml or hygromycin at 0.5 mg/ml. Individual clones were obtained by selection in limiting dilution in 96-well plates as described (29).…”

“…Are Activated by ZNF198-FGFR1-We have previously shown that STATs 1 and 5 are constitutively phosphorylated in cycling Ba/F3 cells transformed to IL-3 independence by the ZNF198-FGFR1 fusion (29). To further characterize STAT activation by ZNF198-FGFR1, we investigated the tyrosine phosphorylation of all six STAT proteins after 24 h of IL-3 withdrawal in three transformed clones.…”

Targeting FGFR3 in multiple myeloma: inhibition of t(4;14)-positive cells by SU5402 and PD173074

“…3;q32) is associated with 10-20% of cases of multiple myeloma (MM) and is believed to dysregulate the expression of two genes, MMSET on the der(4) and FGFR3 on the der (14). 1 At present, it is not known whether deregulation of either or both of these genes is the critical pathogenetic consequence of the t(4;14), but a role for FGFR3 is suggested by several lines of evidence.…”

“…The presence of the t(4;14) was confirmed by fluorescence in situ hybridization using standard procedures. Control cell lines were all t(4;14)-negative: U266 (MM), Ramos and Daudi (Burkitt lymphoma), BaF3/ZNF198-FGFR1 (Ba/F3 transformed by ZNF198-FGFR1 to IL-3 independence), 14 BaF3/BCR-ABL (Ba/F3 cell line transformed by BCR-ABL to IL-3 independence; kindly provided by JV Melo, London). Cells were maintained in tissue culture medium consisting of RPMI 1640 supplemented with 10% foetal calf serum (Gibco BRL) plus 1% penicillin/streptomycin (Gibco BRL) and grown in a humidified incubator maintained at 371C and 5% CO 2 throughout.…”