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

The 8p11 myeloproliferative syndrome is an aggres-sive disorder caused by FGFR1 fusion proteins resulting from a subset of acquired translocations that target chromosome band 8p11. These chimeric proteins have constitutive FGFR1 tyrosine kinase activity and are be-lieved to deregulate hemopoietic development in a manner analogous to BCR-ABL in chronic myeloid leukemia. Here we have studied the role of STAT proteins in transformation mediated by the most common of these fusions, ZNF198-FGFR1. We found that STATs 1, 3, and 5 were activated constitutively in ZNF198-FGFR1-trans-formed Ba/F3 cells and that STATs 2, 4, and 6 were also tyrosine-phosphorylated. Induction of dominant negative STAT mutants showed that activation of STAT5, but not STATs 1 or 3, was essential for the anti-apoptotic effect of ZNF198-FGFR1 and that STAT5 activation is essential for the elevated levels of BclXL in transformed cells. STAT5 activation was also shown to be required for continued cell cycle progression of BaF3/ZNF198-FGFR1 cells in conditions of cytokine deprivation and for up-regulation of the DNA repair protein Rad51. These findings suggest a critical role of STAT5 activation in transformation mediated by ZNF198-FGFR1. Chromosomal translocations that disrupt

The 8p11 myeloproliferative syndrome is an aggressive disorder caused by FGFR1 fusion proteins resulting from a subset of acquired translocations that target chromosome band 8p11. These chimeric proteins have constitutive FGFR1 tyrosine kinase activity and are believed to deregulate hemopoietic development in a manner analogous to BCR-ABL in chronic myeloid leukemia. Here we have studied the role of STAT proteins in transformation mediated by the most common of these fusions, ZNF198-FGFR1. We found that STATs 1, 3, and 5 were activated constitutively in ZNF198-FGFR1-transformed Ba/F3 cells and that STATs 2, 4, and 6 were also tyrosine-phosphorylated. Induction of dominant negative STAT mutants showed that activation of STAT5, but not STATs 1 or 3, was essential for the anti-apoptotic effect of ZNF198-FGFR1 and that STAT5 activation is essential for the elevated levels of BclXL in transformed cells. STAT5 activation was also shown to be required for continued cell cycle progression of BaF3/ZNF198-FGFR1 cells in conditions of cytokine deprivation and for up-regulation of the DNA repair protein Rad51. These findings suggest a critical role of STAT5 activation in transformation mediated by ZNF198-FGFR1.
Chromosomal translocations that disrupt FGFR1 are associated with the disease known as the 8p11 myeloproliferative syndrome (EMS), 1 a stem cell disorder associated with eosinophilia, lymphadenopathy, and rapid progression to acute leukemia (1,2). The most common translocation in EMS is the t (8,13), which results in the fusion of the N-terminal half of ZNF198 to the entire catalytic domain of FGFR1 (3)(4)(5)(6). ZNF198-FGFR1 is a constitutively active tyrosine kinase that transforms growth factor-dependent cell lines to factor independence (7)(8)(9). Several other translocations have been identified that fuse different partner genes to FGFR1 (1); notable among these is BCR-FGFR1, which is associated with a disease that more closely resembles BCR-ABL-positive chronic myeloid leukemia than EMS (8,10).
Definition of signal transduction pathways that are required for transformation by fusion tyrosine kinases is critical to the development of novel targeted therapies. Fusion tyrosine kinases share the ability to activate members of the STAT family of latent cytoplasmic transcription factors, and it has been suggested that STAT activation is a critical event in transformation mediated by oncogenic proteins such as BCR-ABL, TEL-JAK2, and TEL-PDGFRB (11)(12)(13)(14)(15)(16)(17). Using murine models, STAT5 has been shown to be essential for hematological disease induced by TEL-JAK (18) but not BCR-ABL (19), indicating that different fusion tyrosine kinases have distinct modes of action. STAT5 is activated by a wide variety of cytokines (20,21) and has, for example, been shown to be essential for fetal red blood cell production by rescuing committed progenitors from apoptosis, at least in part by direct induction of BclXL (22).
Constitutive activation of STAT3 also occurs in many cancers (23,24). STAT3 is normally activated by diverse ligands, including epidermal growth factor, platelet-derived growth factor, IL-6, oncostatin M, and leukemia inhibitory factor; a role for STAT3 activation in suppression of apoptosis has also been suggested (25)(26)(27). In contrast, activation of STAT1 appears to be associated with negative regulation of cell growth, e.g. STAT1-mediated down-regulation of chondrocyte proliferation in forms of dwarfism that result from activating mutations in FGFR3 (28).
In this study we have investigated the importance of STAT activation in transformation mediated by the ZNF198-FGFR1. We have also characterized the relative transforming properties of ZNF198-FGFR1, BCR-ABL, and BCR-FGFR1.
Transfections-Exponentially growing Ba/F3 cells were washed and resuspended in phosphate-buffered saline. 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).
Measurement of Factor-independent Survival and Proliferation-Factor-independent growth was analyzed immediately after establish-ment of G418-resistant clones in 96-well plates. Clones were replated into 24-well plates in the absence of IL-3 to determine the percentage of clones that survived without this growth factor. Surviving clones were replated into 12-well plates to determine the percentage of clones that proliferated without IL-3. FGFR1-expressing clones were maintained in media without IL-3 with the addition of acidic human fibroblast growth factor (First Link, West Midlands, UK) at 10 ng/ml and heparin (Sigma) at 2 mg/ml.
Induction of Dominant Negative STATs-To express inducible dn STATs, the Lac Switch II inducible expression system was used (Stratagene, La Jolla, CA). Ba/F3 cells and IL-3-independent ZNF198-FGFR1/BaF3 clones were each co-transfected with the Lac repressor pCMV-LacI and either HA-dnSTAT1, HA-dnSTAT3, or HA-dnSTAT5. Clones were selected by culture with hygromycin at limiting dilution. The POPRSV1 expression vector contains the Rous sarcoma virus promoter linked to the Escherichia coli lactose operon, and expression of target DNA is suppressed by the lactose repressor. Addition of isopropyl-␤-D-thiogalactoside at 0.5 mM to the culture media causes the release of Lac-R from the lactose operon and transcription of target DNA (30).
Apoptosis and Cell Cycle Assays-The percentage of dead cells was measured by Trypan blue exclusion, and the percentage of apoptotic cells was measured by analysis of the DNA content by flow cytometry. Apoptotic cells were defined as those with a less than 2n DNA content. Cell cycle distribution was analyzed by the same procedure. After specific culture conditions (growth in the absence of exogenous IL-3 and/or in 0.5% fetal calf serum), 2 ϫ 10 6 cells were fixed in 70% ethanol for 30 min and then incubated in 1 ml of phosphate-buffered saline containing 1 mg of DNase-free RNase (Sigma) and propidium iodide (Sigma) at 50 g/ml for 30 min. DNA content was then analyzed by flow cytometry (FACscan; BD Biosciences).
For immunoprecipitations, total cell lysates from 2 ϫ 10 7 cells were mixed with 2 ml of RIPA buffer (10 mM Tris, pH 8, 150 mM NaCl, 1% Triton, 0.1% SDS) plus 2 g of antibody at 4°C overnight, followed by binding to agarose AG beads (Santa Cruz Biotechnology) at 4°C for 2 h. Immunoprecipitates were washed three times in RIPA buffer plus protease and phosphatase inhibitors and then boiled in 2ϫ loading buffer.
Electrophoretic Mobility Shift Assay (EMSA) Analysis-Nuclear extracts were prepared by suspending cell pellets in 500 l of hypotonic buffer (20 mM Hepes, pH 7.9, 0.2% Nonidet P-40, 0.1 mM EDTA, 1 mM dithiothreitol) plus protease and phosphatase inhibitors as above. After a 10-min incubation on ice, nuclei were recovered by centrifugation at 1000 ϫ g for 5 min. Nuclei were then extracted with gentle rocking on ice in 150 l of high salt buffer (20 mM Hepes, pH 7.9, 0.2% Nonidet P-40, 0.4 M NaCl, 13.3% glycerol, 1mM dithiothreitol) plus protease and phosphatase inhibitors. Lysates were then clarified by centrifugation at 1300 ϫ g for 5 min, and the supernatant was recovered as the nuclear extract. The extracts were rapidly frozen at Ϫ70°C. An aliquot was taken and used to determine the protein concentration.
Nuclear extracts (10 g for each reaction) were incubated with 5 fmoles of 32 P end-labeled probe containing a specific STAT binding sequence in 4% Ficoll, 0.5 g/ml bovine serum albumin, 20 mM Hepes, pH 7.6, 50 mM NaCl, 5 mM MgCl 2, 0.5 mM spermidine, 50 g/ml poly(dI-dC) for 30 min on ice. For supershift analysis the cell extract in binding buffer was incubated with specific antibody on ice for 30 min before addition of labeled probe. For specific competition analysis, nuclear extracts were preincubated with 500 fmoles of unlabeled probe for 30 min prior to incubation with labeled probe. Samples were separated on a non-denaturing 5% gel in 0.2ϫ TBE (1ϫ TBE is 50 mM Tris borate, 1 mM EDTA). Gels were dried and visualized by autoradiography.
For analysis of STAT5 binding activity, the Ϫ105 probe from the ␤-casein promoter was used (sense sequence 5Ј-AGATTTCTAGGAAT-TCAATCC-3Ј) (31). For analysis of STAT1 and STAT3 binding the SIE m67 probe derived from the c-fos promoter was used (sense sequence 5Ј-CATTTCCCGTAAATC-3Ј) (32). For analysis of binding to the BCLXL promoter, a region derived from the mouse BCLXL gene was used (sense sequence, 5Ј-TTTGGAGGGGAACATTTCGGAGAAAAG-3Ј) (33). All oligos were annealed to their corresponding antisense sequence prior to analysis.

STATs 1, 2, 3, 4, 5, and 6
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. STAT activation was compared with parental Ba/F3 cells treated in the same way. Cell lysates were immunoprecipitated with a specific STAT antibody; half the sample was probed with the same STAT antibody to check for loading and expression levels, and the other half was probed with 4G10 (antiphosphotyrosine antibody). In cells transformed by the fusion, but not in control Ba/F3 cells, tyrosine phosphorylation of each STAT protein was detected (Fig. 1).
Constitutive Binding of STATs 1, 3, and 5 to Cognate DNA Sequences Occurs in Ba/F3 Cells Transformed by the ZNF198- FGFR1 Fusion-Activation of STATs 1, 3, and 5 has been associated with transformation induced by BCR-ABL (12,13), and therefore we focused on the activity of these proteins. To determine whether STAT 1, 3, or 5 tyrosine phosphorylation induced functional activation of DNA binding, we carried out EMSAs. Specific activation of STAT1 and STAT3 was assessed by using the high affinity m67sie sequence derived from the c-fos promoter. Three different complexes, which correspond to STAT1 homodimers, STAT1/3 heterodimers, and STAT 3 homodimers, respectively (32), have been characterized to form with this probe. Nuclear extracts from parental Ba/F3 cells that had been grown continuously in the presence of IL-3 or deprived of IL-3 for 24 h were compared with ZNF198-FGFR1-transformed cells that had been treated in the same way. We found a very low level of STAT1 or STAT3 binding in extracts from parental Ba/F3 cells but strong binding in transformed cells. Competition with excess cold m67sie probe confirmed that the complexes observed are specific. The top two complexes were confirmed to contain STAT3 by supershifts induced by STAT3 antibody. Addition of STAT1 antibody induced a reduction in the complexes with STAT1 homo-and heterodimers. (Fig. 2A). Thus, tyrosine phosphorylation of STATs 1 and 3 in fusion-transformed cells confers DNA binding ability.
STAT5 binding activity was analyzed in a similar way using the ␤-casein probe. As shown on Fig. 2B, STAT5 binding was very weak in Ba/F3 cells but strong in transformed cells. Supershifting of the complex with STAT5 antibody confirmed that the complexes did indeed contain STAT5, and competition with excess unlabeled probe confirmed that the binding was specific. This experiment confirms activation of STAT5 by ZNF198-FGFR1.
ZNF198-FGFR1 Expression Blocks Apoptosis Following IL-3 or Serum Deprivation-To assess the ability of ZNF198-FGFR1 expression to block apoptosis induced by cytokine deprivation, we took three G418-resistant clones (C1-C3) and analyzed the percentage of apoptotic cells under different conditions. ZNF198-FGFR1-transformed cells were compared with parental Ba/F3 cells that were growing continuously in IL-3 or had been starved of IL-3 or IL-3 and serum for 24 or 48 h. The percentage of apoptotic cells was measured by FACS analysis (Fig. 3A), and the numbers of dead cells were counted by Trypan blue staining (not shown). As expected, we found a significant increase in apoptosis in parental Ba/F3 cells after 24 h of IL-3 withdrawal, which correlated with an increase in cell death. In contrast, cells transformed by the fusion had a low level of apoptosis and death after both 24 h (Fig. 3A) and 48 h (not shown) of IL-3 or serum withdrawal. Thus, expression of the ZNF198-FGFR1 fusion, which was expressed at similar levels in the three clones (Fig. 3B), protects transformed cells from apoptosis due to cytokine deprivation.
Effects of ZNF198-FGFR1 Expression on BclXL Protein Levels-To investigate further the signaling mechanisms involved in the resistance to apoptosis observed in ZNF198-FGFR1transformed cells, we compared protein levels of the BCL-2 family members BclXL, Bcl2, and Mcl-1 by immunoblotting. Levels of these proteins were compared in parental Ba/F3 cells and transformed cells under the following conditions: cycling in the presence of IL-3, 24 and 48 h following IL-3 withdrawal or the same time points following serum and IL-3 withdrawal. We found that in parental Ba/F3 cells, down-regulation of BclXL protein levels occurred after 24 h of IL-3 withdrawal, with or without serum, but that the levels of other BCL2 family members were unaffected (Fig. 3C). In contrast, levels of BclXL protein in cells transformed by the fusion remained elevated after withdrawal of either IL-3 or serum and IL-3 at both 24 h (Fig. 3C) and 48 h (not shown).
Inducible Expression of Dominant Negative STATs in Cells Transformed by the ZNF198-FGFR1 Fusion-To investigate the function of specific STAT activation in transformation, proliferation, and resistance to apoptosis conferred by the fusion, we prepared several stable clones of ZNF198-FGFR1transformed cells that inducibly expressed dominant negative forms of HA-tagged STAT1, STAT3, and STAT5, plus Ba/F3 cells that inducibly expressed dnSTAT5. Although stable transfectants expressing dnSTAT1 and dnSTAT3 were easily obtained in media without IL-3, we failed to obtain clones expressing dnSTAT5. We suspected a cytotoxic effect of low levels of dnSTAT 5 under these conditions as a consequence of leaky transcription. Clones inducibly expressing dnSTAT 5 were, however, readily obtained in the presence of IL-3.
Western blot analysis of HA-immunoprecipitated proteins confirmed that the addition of 0.5 mM isopropyl-␤-D-thiogalactoside to the culture media lead to the induction of dnSTAT1, dnSTAT3, and dnSTAT5 with maximum expression 24 h after induction. A slight variation in levels of expression of the dnSTATs was observed in different clones, and one dnSTAT1 clone was found to express protein without induction (Fig. 4). To evaluate the inhibitory effects of these dnSTATs on the constitutive binding to cognate DNA sequences observed with extracts from cells transformed by ZNF198-FGFR1, we carried out EMSA analysis. As before, nuclear extracts were isolated from parental Ba/F3 cells, Ba/F3 cells with induced dnSTAT5, and from ZNF198-FGFR1-transformed cells with induction of either dnSTAT1, dnSTAT3, or dnSTAT5. Extracts were prepared from cells growing with IL-3, after IL-3 deprivation for 24 h, and with serum and IL-3 deprivation for 24 h. We found that induction of dnSTAT1 and dnSTAT3 greatly decreased the binding of these STATs to the m67sie probe observed in ZNF198-FGFR1-transformed cell lysates under all conditions (Fig. 5A). Similarly, induction of dnSTAT5 greatly decreased the complex formed with the ␤-casein probe but did not completely ablate binding (Fig. 5B). These experiments confirm that induction of the specific dnSTATs do indeed function to inhibit DNA binding of STATs activated by the ZNF198-FGFR1 fusion.

Dominant Negative STAT5 Induction Increases Cell Apoptosis on IL-3 and Serum Withdrawal of Ba/F3 Cells Transformed by ZNF198-FGFR1-
To investigate the effects of inhibiting activation of specific STAT proteins on apoptosis of transformed Ba/F3 cells, we compared the proportion of cells that had a subdiploid DNA content or were dead in cells with or without induction of dnSTAT1, dnSTAT3, or dnSTAT5. We found that induction of either dnSTAT1 or dnSTAT3 had little or no discernable effect on the proportion of apoptotic cells in ZNF198-FGFR1-transformed cells after IL-3 or serum withdrawal (not shown). In contrast, induction of dnSTAT5 was found to greatly increase the percentage of apoptosis following both IL-3 or serum and IL-3 (Fig. 6A) in ZNF198-FGFR1transformed clones that expressed comparable levels of the fusion protein. These results indicate that constitutive activation of STAT5 by ZNF198-FGFR1 plays an essential role in the observed resistance to apoptosis. Substantial levels of apoptotic cells were also seen in Ba/F3 cells expressing dnSTAT5, consistent with previous studies demonstrating that STAT5 plays an important role in IL-3-mediated signal transduction (31).
ZNF198-FGFR1-mediated STAT5 Activation Is Essential for the Elevated Levels of BclXL Protein in Transformed Cells-We used dnSTAT1, dnSTAT3, and dnSTAT5 induction to determine which STAT participates in the activation of BclXL protein expression observed in cells transformed by the ZNF198-FGR1 fusion. We found that induction of either dnSTAT1 or dnSTAT3 had no effect on the elevated levels of BclXL protein found in cells expressing the fusion after serum or IL-3 deprivation. Protein levels of other BCL2 family members examined were also unaffected by dnSTAT1 or dnSTAT3 induction. In contrast, induction of dnSTAT5 caused a significant decrease in the level of BclXL protein expressed in fusion-transformed cells under all conditions (Fig. 6B). This is consistent with previous studies demonstrating that BCLXL is a direct transcriptional target of STAT5 (22). Protein levels of other BCL2 family members were not affected by dnSTAT5 expression (Fig.  6B), and no clear reduction in BclXL levels was seen on induction of STAT5 in Ba/F3 cells without the fusion (Fig. 6C). The decrease in BclXL protein levels due to dnSTAT5 induction correlates with the increase in apoptotic cell death observed. This suggests that activation of BclXL expression by the ZNF198-FGFR1 fusion via constitutive STAT5 expression is important in conferring the observed resistance to apoptosis.
Activated STAT5 Binds to the BclXL Promoter in Cells Transformed by ZNF198-FGFR1, and dnSTAT5 Induction In-hibits This Binding-We used EMSA to confirm that dnSTAT5 reduces BclXL levels by blocking the binding of STAT5 to the BCLXL promoter. As expected the 32 P-labeled probe, which was derived from the murine BCLXL promoter, formed a complex in ZNF198-FGFR1-transformed cells, but this complex was markedly reduced in Ba/F3 cells cultured either in the presence or absence of IL-3 (Fig. 7). Preincubation of nuclear extracts from transformed cells with 100-fold molar excess of unlabeled probe prevented formation of this complex, confirming specificity. The complex was supershifted by anti-STAT5  antibody, confirming that this BCLXL probe binds STAT5 specifically. We then investigated the effect of induction of either dnSTAT1, dnSTAT3, or dnSTAT5 on formation of the complex with the BCLXL probe. Induction of dnSTAT5 in cells transformed by the fusion caused a significant reduction in the bound complex under all conditions (Fig. 7). No effect on this complex formation was observed with induction of dnSTAT1 or dnSTAT3 (data not shown), and no or minimal effect was observed on induction of dnSTAT5 in Ba/F3 cells (Fig. 7). These results confirm that ZNF198-FGFR1-mediated STAT5 activation causes transcriptional activation of BCLXL by direct binding of STAT5 to the BCLXL promoter.
STAT5 Activation Is Essential for Continued Cell Cycle Progression in ZNF198-FGFR1-transformed Cells-We sought to investigate whether STAT1, STAT3, or STAT5 activation affected cell cycle progression of Ba/F3 cells transformed by the fusion. Initially cell cycle progression of parental Ba/F3 cells, either growing with IL-3, starved of IL-3 for 24 or 48 h, or starved of serum and IL-3 for 24 or 48 h, was compared with that of similarly treated cells transformed by the ZNF198-FGFR1 fusion. Cell cycle status was examined by FACS analysis of cells after propidium iodide staining. We found that parental Ba/F3 cells arrested in G 0 /G 1 following either IL-3 or serum deprivation for 24 h (lack of cells in S or G 2 /M phase; Fig.  8). Cells transformed by the fusion showed no G 0 /G 1 arrest after either IL-3 or serum withdrawal for 24 or 48 h but continued to progress through the cell cycle under all conditions. The effect of induction of either dnSTAT1, dnSTAT3, or dnSTAT5 in cells transformed by the fusion was then examined. We found that induction of neither dnSTAT1 nor dnSTAT3 affected the continued cell cycling of transformed cells in conditions of low cytokine or serum deprivation (data not shown). In contrast, induction of dnSTAT5 caused a dramatic block in cell cycle progression. Hardly any cells were observed to progress to S or G 2 /M phase with dnSTAT5 induction and the number of cells in the sub-G 1 peak dramatically increased (Fig. 8). These results indicate that STAT5 activation by the ZNF198-FGFR1 fusion plays an important role in causing the continued cell cycle progression under conditions of low cytokine or serum in transformed cells.
ZNF198-FGFR1-mediated STAT5 Activation Also Results in Elevated Levels of Rad51-Previous studies with cells transformed by BCR-ABL indicated that STAT5 activation mediates up-regulation of Rad51, a protein involved in the repair of double-stranded DNA breaks (34). We therefore investigated whether Rad51 protein levels were elevated in cells transformed by ZNF198-FGFR1. As shown on Fig. 9, we found that levels of Rad51 were elevated in cells transformed by the fusion compared with parental Ba/F3 cells either in the presence or absence of IL-3. Induction of dnSTAT5 (Fig. 9) in ZNF198-FGFR1-transformed cells, but not induction of dnSTAT1 or dnSTAT3 (not shown), caused a reduction in the levels of Rad51, whereas levels of Rad51 were comparable in parental Ba/F3 cells with or without induction of dnSTAT5 (Fig. 9). This indicates a role for STAT5 activation in the up-regulation of Rad51 protein levels by ZNF198-FGFR1.
Comparison of the Transforming Abilities of ZNF198-FGFR1, BCR-ABL, and BCR-FGFR1-The degree with which fusion tyrosine kinases transform Ba/F3 cells is a measure of their oncogenicity. We have sought to determine whether there are any differences in the transforming potential of ZNF198-FGFR1, BCR-FGFR1, and BCR-ABL in Ba/F3 cells. Ba/F3 cells were independently transfected with the three fusion genes, with normal FGFR1, and with vector only as a negative control. G418-resistant clones were isolated by limiting dilution in 96well plates and replated into media without IL-3. Surviving clones were replated again, and the percentage of G418-resistant clones that (i) survived and (ii) proliferated was calculated for each fusion. We found that 70% of ZNF198-FGFR1 clones survived, but only 6 -7% were transformed to IL-3-independent growth. In contrast, for BCR-ABL 98% of G418-resistant cells survived and 65% proliferated. The difference in transforming strength between these two fusions is not a consequence of the different kinase domains and associated regulatory regions because the great majority of cells transfected with full-length FGFR1 survived and proliferated in the presence of aFGF plus heparin. Finally, cells transfected with BCR-FGFR1 showed a pattern more similar to BCR-ABL than ZNF198-FGFR1 with 95% survival and 75% proliferation (Fig. 10). DISCUSSION The abnormal expansion of hematopoietic progenitor cells in EMS could be mediated partly by inhibition of apoptosis and partly by promotion of proliferation, both as a consequence of deregulated FGFR1 signaling. We have shown that Ba/F3 cells transformed by ZNF198-FGFR1 are resistant to apoptosis on both serum and cytokine withdrawal and continue to progress through the cell cycle under low serum or cytokine conditions. The resistance to apoptosis in fusion-transformed cells correlates with elevated levels of the anti-apoptotic protein BclXL. Identification of the critical signaling pathways downstream of FGFR1 fusion proteins that are responsible for these apoptotic and proliferative defects is important because it will aid the identification of specific inhibitory drugs for treatment of patients with EMS.
We and others have previously shown that several signaling pathways associated with the promotion of proliferation and the inhibition of apoptosis are activated by FGFR1 fusion proteins, including pathways that involve mitogen-activated protein kinase, phospholipase C ␥, phosphatidylinositol 3 kinase, and STATs (7-9, 18, 35). Here we have focused on the role of STAT proteins. We have shown that all six STATs are phosphorylated in BaF3/ZNF198-FGFR1 cells. This contrasts with a previous study that showed phosphorylation of STATs 1, 3, FIG. 7. Binding of STAT5 to the BclXL promoter in ZNF198-FGFR1-transformed cells and inhibition of this binding by induction of dnSTAT5. Nuclear extracts were analyzed by EMSA using a region of the BclXL promoter containing a STAT5 binding sequence as a probe. Complexes formed were compared with those formed with nuclear lysates from Ba/F3 cells transformed by ZNF198-FGFR1 and with lysates from ZNF198-FGFR1-transformed cells and Ba/F3 cells with induction of dnSTAT5, treated in the same way. Lanes are labeled as for Fig. 6; specific STAT 5 complexes are indicated with an arrow. The supershifted complex is indicated by a triangle. and 5, but not STATs 4 and 6, in ZNF198-FGFR1-transfected 293 cells (9). Lack of STAT4 and STAT6 phosphorylation was observed even in 293 cells that were engineered to express high levels of these proteins. One possible explanation for this difference could be that phosphorylation of STAT4 and STAT6 is not mediated directly by ZNF198-FGFR1 but rather through an intermediary that is expressed in Ba/F3 cells but not 293 cells.
We focused our analysis on STATs 1, 3, and 5 because these proteins have been shown to play an important role in hemo- poietic cells and also for transformation by other tyrosine kinase fusion genes (18,23,24). We show that phosphorylation of STATs 1, 3, and 5 in cells transformed by ZNF198-FGFR1 is, as expected, associated with an increase in DNA binding to their cognate sequences. Using inducible expression of dnSTATs, we have shown that STAT5, but not STAT1 or STAT3, has a dramatic effect on both the resistance to apoptosis and the ability to proliferate in low cytokine or serum conditions. The induction of dnSTAT5 in ZNF198-FGFR1-transformed cells correlated with a decrease in expression levels of BclXL, but not other BCL2 family members, indicating that STAT5 activation is essential for the elevated levels of BclXL. Furthermore, the correlation of increased apoptosis with decreased BclXL levels strongly indicates that resistance to apoptosis in ZNF198-FGFR1-transformed cells is mediated via the up-regulation of BclXL. Previous reports have described activation of STAT3 as being important for the resistance to apoptosis conferred by other oncogenes (23). However, we found no increase in apoptosis in transformed cells after dnSTAT3 induction, indicating that activation of STAT5 by the ZNF198-FGFR1 fusion is the primary signaling pathway involved in conferring resistance to apoptosis in these cells.
Increased proliferation due to continued cell cycling in conditions of low serum or cytokines is characteristic of transformed cells. Here we show that Ba/F3 cells transformed by the ZNF198-FGFR1 fusion continue to progress through the cell cycle after withdrawal of either IL-3 or both IL-3 and serum. Parental Ba/F3 cells arrest in G 0 /G 1 under these conditions. Induction of dnSTAT5 but not dnSTATs1 or 3 inhibited the ability of ZNF198-FGFR1 cells to progress through the cell cycle. This indicates that activation of STAT5 by the fusion is the principal mechanism involved in conferring the ability to proliferate under low cytokine or serum conditions. This effect is likely to involve the cyclin D family, members of which are known STAT5 targets (30).
Cells transformed by BCR-ABL and some other fusion tyrosine kinases have been reported to have levels of Rad51 that are elevated by a STAT5-dependent mechanism (34). Rad51 plays a key role in double strand break repair by homologous recombination, and it has been suggested that overexpression of this protein could contribute to both genomic instability and drug resistance (34). Here we have shown that protein levels of Rad51 are also elevated in cells transformed by ZNF198-FGFR1 under conditions of cytokine withdrawal and that these elevated levels are dependent on activation of STAT5 but not STATs 1 or 3.
We found that ZNF198-FGFR1 transforms Ba/F3 cells relatively weakly compared with BCR-ABL and BCR-FGFR1. The precise reasons for this difference are under investigation, but it is striking that the transforming ability of these three fusions in vitro appears to correlate with patient phenotype in that BCR-ABL and BCR-FGFR1 are both associated with a chronic myeloid leukemia-like disease, whereas ZNF198-FGFR1 is associated with a related but distinctive disease, EMS. Analysis of other fusions in a similar manner should help to determine whether there is a correlation between the oncogenicity of tyrosine kinase fusion proteins and patient phenotype.