Mass spectroscopy identifies the splicing-associated proteins, PSF, hnRNP H3, hnRNP A2/B1, and TLS/FUS as interacting partners of the ZNF198 protein associated with rearrangement in myeloproliferative disease

ZNF198 is fused with FGFR1 in an atypical myeloproliferative disease that results in constitutive activation of the kinase domain and mislocalization to the cytoplasm. We have used immunoprecipitation of a GFP-tagged ZNF198 combined with MALDI-TOF mass spectroscopy to identify interacting proteins. P splicing factor (PSF) was identified as one of the proteins and this interaction was confirmed by Western blotting. Other proteins identified were the spliceosomal components hnRNP A2/B1, hnRNP H3, and TLS/FUS. PSF is also known to interact with PTB, another member of the hnRNP family of proteins, and we further demonstrated that PTB interacts with ZNF198. The interaction between TLS/FUS and ZNF198 was confirmed using Western blot analysis. In 293 cells expressing the ZNF198/FGFR1 fusion protein, neither PSF nor PTB binds to the fusion protein, possibly because of their differential localization in the cell.     

Critical role of STAT5 activation in transformation mediated by ZNF198-FGFR1

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.     

Phosphorylation of the SSBP2 and ABL proteins by the ZNF198‐FGFR1 fusion kinase seen in atypical myeloproliferative disorders as revealed by phosphopeptide‐specific MS

The ZNF198‐fibroblast growth factor receptor‐1 (FGFR1) fusion kinase is a constitutively activated tyrosine kinase associated with a specific atypical myeloproliferative disease. The chimeric protein localizes to the cytoplasm, unlike the wild type FGFR1 receptor kinase, and presumably inappropriately phosphorylates specific targets as part of the oncogenic signaling cascade. Other than known targets of the FGFR1 kinase itself, few specific targets of ZNF198‐FGFR1 have been identified. Using a genetically engineered HEK 293 cell system, we have identified proteins that are specifically phosphorylated in the presence of the fusion kinase using anti‐phosphotyrosine immunoprecipitation and MS. Compared with 293 cells expressing exongenous wild type FGFR1, ZNF198‐FGFR1 is associated with phosphorylation of several proteins including SSBP2, ABL, FLJ14235, CALM and TRIM4 proteins. The specificity of the phosphorylation events in the SSBP2 and ABL proteins, which have previously been implicated in leukemogenesis, was further confirmed independently using immunoprecipitation with protein‐specific antibodies and Western blotting. The MS analysis also identified the phosphorylation events in the ZNF198 moiety in the chimeric protein that might be related to its function. These studies identify the intersection of several different leukemia‐related pathways in the development of this myeloproliferative disorder and provide new insights into the substrates of FGFR1 under defined conditions.     

Oncogenic fusion protein FGFR2-PPHLN1: Requirements for biological activation,and efficacy of inhibitors

Aim of studyChromosomal translocations such as t(10;12)(q26,q12) are associated with intrahepatic cholangiocarcinoma, a universally fatal category of liver cancer. This translocation creates the oncogenic fusion protein of Fibroblast Growth Factor Receptor 2 joined to Periphilin 1. The aims of this study were to identify significant features required for biological activation, analyze the activation of downstream signaling pathways, and examine the efficacy of the TKIs BGJ398 and TAS-120, and of the MEK inhibitor Trametinib.MethodsThese studies examined FGFR2-PPHLN1 proteins containing a kinase-dead, kinase-activated, or WT kinase domain in comparison with analogous FGFR2 proteins. Biological activity was assayed using soft agar colony formation in epithelial RIE-1 cells and focus assays in NIH3T3 cells. The MAPK/ERK, JAK/STAT3 and PI3K/AKT signaling pathways were examined for activation. Membrane association was analyzed by indirect immunofluorescence comparing proteins altered by deletion of the signal peptide, or by addition of a myristylation signal.ResultsBiological activity of FGFR2-PPHLN1 required an active FGFR2-derived tyrosine kinase domain, and a dimerization domain contributed by PPHLN1. Strong activation of canonical MAPK/ERK, JAK/STAT3 and PI3K/AKT signaling pathways was observed. The efficacy of the tyrosine kinase inhibitors BGJ398 and TAS-120 was examined individually and combinatorially with the MEK inhibitor Trametinib; heterogeneous responses were observed in a mutation-specific manner. A requirement for membrane localization of the fusion protein was also demonstrated.Concluding statementOur study collectively demonstrates the potent transforming potential of FGFR2-PPHLN1 in driving cellular proliferation. We discuss the importance of sequencing-based, mutation-specific personalized therapeutics in treating FGFR2 fusion-positive intrahepatic cholangiocarcinoma.     

The Genomic Structure of ZNF198 and Location of Breakpoints in the t(8;13) Myeloproliferative Syndrome

The t(8;13)(p11;q12) is the most common translocation associated with the 8p11 myeloproliferative syndrome and results in an identical mRNA fusion between ZNF198 at 13q12 and FGFR1 at 8p11 in all cases thus far reported. ZNF198 is a widely expressed gene that is predicted to encode a 1377-amino-acid protein with five Zn finger-related motifs known as MYM domains. To determine the genomic DNA structure of ZNF198, we employed bubble PCR from PAC clones with a panel of gene-specific primers. Sequencing of these products revealed that ZNF198 consists of 26 exons with the initiation codon located in exon 4. The t(8;13) results in a consistent mRNA fusion of ZNF198 exon 17 to FGFR1 exon 9. Notable features of the structure of ZNF198 include three noncanonical GC donor splice sites and the presence of an alternatively spliced intron within exon 4. Amplification of genomic DNA from six t(8;13) patients with primers to ZNF198 exon 17 and FGFR1 exon 9 yielded patient-specific products ranging in size from 500 bp to 2.5 kb, indicating that the positions of the breakpoints in the t(8;13) are tightly clustered. The positions of the six t(8;13) breakpoints were determined and found to be distributed across ZNF198 intron 17 and FGFR1 intron 8 with no apparent subclustering. No consistent sequence motifs, repeats, or topoisomerase II cleavage sites were found at or near the breakpoints. It remains unclear why the t(8;13) translocation breakpoints occur within such small genomic regions, and it is possible that strict ZNF198–FGFR1 coding requirements restrict the positions of the breakpoints.     

ZNF198, a zinc finger protein rearranged in myeloproliferative disease, localizes to the PML nuclear bodies and interacts with SUMO-1 and PML

The ZNF198/FGFR1 fusion gene in atypical myeloproliferative disease produces a constitutively active cytoplasmic tyrosine kinase, unlike ZNF198 which is normally a nuclear protein. We have now shown that the ZNF198/FGFR1 fusion kinase interacts with the endogenous ZNF198 protein suggesting that the function of ZNF198 may be compromised in cells expressing it. Little is currently known about the endogenous function of ZNF198 and to investigate this further we performed a yeast two-hybrid analysis and identified SUMO-1 as a binding partner of ZNF198. These observations were confirmed using co-immunoprecipitation which demonstrated that ZNF198 is covalently modified by SUMO-1. Since many of the SUMO-1-modified proteins are targeted to the PML nuclear bodies we used confocal microscopy to show that SUMO-1, PML and ZNF198 colocalize to punctate structures, shown by immunocytochemistry to be PML bodies. Using co-immunoprecipitation we now show that PML and sumoylated ZNF198 can be found in a protein complex in the cell. Mutation of the SUMO-1 binding site in wild-type ZNF198 resulted in loss of distinct PML bodies, reduced PML levels and a more dispersed nuclear localization of the PML protein. In cells expressing ZNF198/FGFR1, which also lack the SUMO-1 binding site, SUMO-1 is preferentially localized in the cytoplasm, which is associated with loss of distinct PML bodies. Recently, arsenic trioxide (ATO) was proposed as an alternative therapy for APL that was resistant to traditional therapy. Treatment of cells expressing ZNF198/FGFR1 with ATO demonstrated reduced autophosphorylation of the ZNF198/FGFR1 protein and induced apoptosis, which is not seen in cells expressing wild-type ZNF198. Overall our results suggest that the sumoylation of ZNF198 is important for PML body formation and that the abrogation of sumoylation of ZNF198 in ZNF198/FGFR1 expressing cells may be an important mechanism in cellular transformation.     

Negative regulation of hematopoiesis by the fused in myeloproliferative disorders gene product

The t(8;13) translocation, found in a rare and aggressive type of stem cell myeloproliferative disorder, leads to the generation of a fusion protein between the N-terminal gene product of fused in myeloproliferative disorders (FIM)/ZNF198 and the fibroblast growth factor receptor 1 (FGFR1) kinase domain. The chimeric protein was reported to have constitutively activated tyrosine kinase activity. However, little is known about a role of FIM in hematopoietic cell regulation. Here we show that FIM protein is ubiquitously expressed in mouse embryonic tissues but much less in hematopoietic cells. We also show that forced expression of FIM inhibits the emergence of hematopoietic cells in the cultured mouse aorta-gonad-mesonephros (AGM) region on embryonic day (E) 11.5, where definitive hematopoiesis is first found during embryogenesis. These results suggest that the expression level of FIM determines the development of hematopoiesis during mouse ontogeny.     

8p12 Stem Cell Myeloproliferative Disorder: the FOP-Fibroblast Growth Factor Receptor 1 Fusion Protein of the t(6;8) Translocation Induces Cell Survival Mediated by Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase/Akt/mTOR Pathways

The FOP-fibroblast growth factor receptor 1 (FGFR1) fusion protein is expressed as a consequence of a t(6;8) (q27;p12) translocation associated with a stem cell myeloproliferative disorder with lymphoma, myeloid hyperplasia and eosinophilia. In the present report, we show that the fusion of the leucine-rich N-terminal region of FOP to the catalytic domain of FGFR1 results in conversion of murine hematopoietic cell line Ba/F3 to factor-independent cell survival via an antiapoptotic effect. This survival effect is dependent upon the constitutive tyrosine phosphorylation of FOP-FGFR1. Phosphorylation of STAT1 and of STAT3, but not STAT5, is observed in cells expressing FOP-FGFR1. The survival function of FOP-FGFR1 is abrogated by mutation of the phospholipase C gamma binding site. Mitogen-activated protein kinase (MAPK) is also activated in FOP-FGFR1-expressing cells and confers cytokine-independent survival to hematopoietic cells. These results demonstrate that FOP-FGFR1 is capable of protecting cells from apoptosis by using the same effectors as the wild-type FGFR1. Furthermore, we show that FOP-FGFR1 phosphorylates phosphatidylinositol 3 (PI3)-kinase and AKT and that specific inhibitors of PI3-kinase impair its ability to promote cell survival. In addition, FOP-FGFR1-expressing cells show constitutive phosphorylation of the positive regulator of translation p70S6 kinase; this phosphorylation is inhibited by PI3-kinase and mTOR (mammalian target of rapamycin) inhibitors. These results indicate that translation control is important to mediate the cell survival effect induced by FOP-FGFR1. Finally, FOP-FGFR1 protects cells from apoptosis by survival signals including BCL2 overexpression and inactivation of caspase-9 activity. Elucidation of signaling events downstream of FOP-FGFR1 constitutive activation provides insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.     

Overexpression and purification of single zinc finger peptides of human zinc finger protein ZNF191

ZNF191, a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain structural information of zinc finger domain a convenient method for obtaining milligram quantities of each zinc finger peptide of ZNF191 is necessary. Here, we report an Escherichia coli expression system for rapid and high-level expression of zinc finger 3 and zinc finger 4 of ZNF191. The gene of zinc finger 3 or zinc finger 4 was cloned into pET31b vector to allow expression of single zinc finger peptide as a ketosteroid isomerase (KSI) fusion protein. The KSI-single zinc finger fusion protein was overexpressed in the form of inclusion body, which can be purified by washing several times using buffer solutions, and then be cleaved directly by cyanogen bromide to release single zinc finger peptide. The more than 20mg/L yield of single zinc finger peptide was achieved with more than 95% purity by using YM ultrafiltration membranes. Circular dichroism spectra of these two single zinc finger peptides titrated with Zn(2+) ions demonstrate that they have different secondary structures.