stem cell leukemia-lymphoma syndrome usually presents itself as a myeloproliferative disorder (MPD) that evolves to acute myeloid leukemia and/or lymphoma. retrovirus gene (HERV-K) fused in frame to the C-terminal FGFR1 kinase domain name (7). Although the transforming properties of HERV-K-FGFR1 have not been characterized the other four FGFR1 fusion proteins are constitutively active tyrosine kinases and transform Ba/F3 murine hematopoietic cells to IL-3-impartial growth (8-11). In addition expression of ZNF198-FGFR1 results in increased tyrosine phosphorylation of STAT1 and STAT5 in Ba/F3 cells (10) and the FOP-FGFR1 fusion induces cell Riociguat (BAY 63-2521) survival by activating the PLC-γ mitogen-activated protein kinase/extracellular regulated kinase and phosphatidylinositol 3-kinase (PI3K)/protein kinase B/molecular Mouse monoclonal to Caveolin 1 target of rapamycin pathways (11). These findings indicate that activation of FGFR1 Riociguat (BAY 63-2521) tyrosine kinase and its downstream-signaling pathways play an essential role in pathogenesis of MPD induced by distinct FGFR1 fusion proteins. ZNF198 is widely expressed and has two isoforms that contain either 4 or 10 atypical zinc fingers a proline-rich domain name and an acidic domain name. The ZNF198-FGFR1 fusion protein incorporates an intact FGFR1 C-terminal tyrosine kinase domain name fused to N-terminal ZNF198 zinc fingers and proline-rich domains. ZNF198-FGFR1 is usually predominantly cytoplasmic (8) and activated by constitutive oligomerization (9). We report that ZNF198-FGFR1 induces a myeloproliferative phenotype in a murine bone marrow transplant (BMT) assay and the ZNF198 proline-rich domain name is essential for transforming activity and as well as in a patient with ZNF198-FGFR1-associated MPD. Materials and Methods DNA Constructs. The complete ZNF198-FGFR1 cDNA and truncated ZNF198-FGFR1 constructs were generated and subcloned into retroviral vectors MSCV-neoEB and MSCV2.2IRESGFP as described in ref. 9. Cell Cultures Retrovirus Production and Ba/F3 Cell IL-3 Independence Proliferation Assays. Ba/F3 cells were cultured in RPMI medium 1640 with 10% FBS and 1.0 ng/ml IL-3 (R & D Systems). The 293T cells were cultured in DMEM with 10% FBS. The retroviral stocks were generated Riociguat (BAY 63-2521) and the viral titers were determined as described in refs. 15 and 16. For the murine BMT assays the viral titers of all constructs were normalized to 1 1 × 106 infectious models/ml. Ba/F3 cell lines stably expressing ZNF198-FGFR1 variants were generated and IL-3-impartial growth was assayed as described in ref. 17. For cell viability assays 1 × 105 Ba/F3 cells were cultured in 24-well plates with increasing concentrations Riociguat (BAY 63-2521) of PKC412 in the absence of IL-3. The relative cell viability at each experimental time point was determined by using the Celltiter96AQueous One answer proliferation kit (Promega). Western Blotting Riociguat (BAY 63-2521) and RT-PCR. When assayed for phosphorylation levels of different protein factors Ba/F3 cells were either serum starved or in some experiments treated with PKC412 for 4 h before being lysed. The cell extracts were analyzed by enzyme-linked immunoblotting. Antibodies included rabbit antibodies recognizing FGFR1 STAT5b phospho-PI3K-p85 (Tyr-508) (Santa Cruz Biotechnology) mouse 4G10 antiphosphotyrosine antibody (Upstate Biotechnology Lake Placid NY) and rabbit antibodies recognizing phospho-STAT5 (Tyr-694) PLC-γ1 and phospho-PLC-γ1 (Tyr-783) (Cell Signaling Technology Beverly MA). RT-PCR analysis of RNA derived from patient bone marrow samples was performed as described in Riociguat (BAY 63-2521) ref. 3. Murine BMT Assay and PKC412 Treatment of the Animals. The murine BMT assays and drug treatment were performed as described in refs. 18 and 19. Bone marrow cells (1 × 106) transduced with distinct retroviral constructs were injected into the lateral tail veins of lethally irradiated (450 cGy × 2) syngeneic BALB/c recipient mice. For secondary transplantation 1 × 106 spleen cells from primary recipients were injected into sublethally irradiated (450 cGy × 1)..