WT1 Zinc Finger Research Articles

RNA Binding by the KTS Splice Variants of Wilms' Tumor Suppressor Protein WT1.

Wilms' tumor suppressor protein WT1 regulates the expression of multiple genes through binding of the Cys2-His2 zinc finger domain to promoter sites. WT1 has also been proposed to be involved in post-transcriptional regulation, by binding to RNA using the same set of zinc fingers. WT1 has two major splice variants, where the Lys-Thr-Ser (KTS) tripeptide is inserted into the linker between the third and fourth zinc fingers. To obtain insights into the mechanism by which the different WT1 splice variants recognize both DNA and RNA, we have determined the solution structure of the WT1 (-KTS) zinc finger domain in complex with a 29mer stem-loop RNA. Zinc fingers 1-3 bind in a widened major groove favored by the presence of a bulge nucleotide in the double-stranded helical stem. Fingers 2 and 3 make specific contacts with the nucleobases in a conserved AUGG sequence in the helical stem. Nuclear magnetic resonance chemical shift mapping and relaxation analysis show that fingers 1-3 of the two splice variants (-KTS and +KTS) of WT1 form similar complexes with RNA. Finger 4 of the -KTS isoform interacts weakly with the RNA loop, an interaction that is abrogated in the +KTS isoform, and both isoforms bind with similar affinity to the RNA. In contrast, finger 4 is required for high-affinity binding to DNA and insertion of KTS into the linker of fingers 3 and 4 abrogates DNA binding. While finger 1 is required for RNA binding, it is dispensable for binding to consensus DNA sites.

Methods to Identify and Validate WT1-RNA Interaction.

Tumor suppressor protein, Wt1 is a transcription factor that binds to DNA sequence similar to the Early Growth Response gene, EGR1 consensus binding sequence. Biophysical and biochemical validations have shown that the zinc fingers of Wt1 are capable of binding to both DNA and RNA albeit with different binding affinities which potentially is also isoform specific. SELEX based identification of the RNA binding motifs led to the identification of motifs which could not be translated into the in vivo context. With the advent of recent technologies that allow cross-linking of RNA and protein and high throughput sequencing techniques, it is now possible to analyze the in vivo RNA binding interactome of Wt1. This chapter outlines the initial studies that were aimed at addressing the Wt1 RNA interactome and also provides a detailed overview of some of the recent techniques used.

Measuring Equilibrium Binding Constants for the WT1-DNA Interaction Using a Filter Binding Assay.

Equilibrium binding of WT1 to specific sites in DNA and potentially RNA molecules is central in mediating the regulatory roles of this protein. In order to understand the functional effects of mutations in the nucleic acid-binding domain of WT1 proteins and/or mutations in the DNA- or RNA-binding sites, it is necessary to measure the equilibrium constant for formation of the protein-nucleic acid complex. This chapter describes the use of a filter binding assay to make accurate measurements of the binding of the WT1 zinc finger domain to the consensus WT1-binding site in DNA. The method described is readily adapted to the measurement of the effects of mutations in either the WT1 zinc finger domain or the putative binding sites within a promoter element or cellular RNA.

WT1 Protects Leukemic Cells from Cytotoxic Replicative Stress

BACKGROUND:The transcription factor WT1 is a “pan-leukemic marker”. WT1 is overexpressed in a significant number of AML, CML, ALL, and MDS cells. Moreover, the persistence of WT1 expression after chemotherapy indicates residual leukemic stem cells. Hydroxyurea can decrease the white blood cell count in leukemic patients. Hydroxyurea inhibits ribonucleotide reductase and consequently depletes the cellular dNTP pool. This process induces replicative stress and activates the intra-S phase checkpoint. The prolonged arrest of DNA replication leads to double strand DNA breaks and apoptosis. The aim of our study was to investigate whether WT1 has an impact on the survival of AML and CML cells treated with hydroxyurea. Furthermore, we analyzed whether replicative stress affects WT1 expression. We also addressed whether clinically relevant drugs modulating WT1 influence apoptosis, autophagy, and DNA damage signaling in leukemic cells.METHODS:We used human AML and CML cells (MV4-11, RS4-11, NB4, HEL, K562) and primary AML cells with various genetic lesions (FLT3-ITD, MLL-AF4, PML-RARα, JAK2V617F, BCR-ABL; p53 mutant or null) or a complex karyotype. Cells were exposed to 0,5 mM hydroxyurea and analyzed by FACS, Western Blot and mRNA expression analyses. RNAi with morpholinos targeting the WT1 mRNA as well as the tyrosine kinase inhibitor imatinib (0,1-1 µM) and epigenetic modifiers of the histone deacetylase inhibitor family (HDACi: 1,5-5 µM entinostat and 10-100 nM panobinostat) were used to decrease WT1 levels.RESULTS:1.The stability of WT1 is causally linked to the survival of leukemic cells undergoing replicative stress. Hydroxyurea-treated leukemic cells retaining WT1 (K562 and RS4-11 cells) accumulate in S phase and survive. However, cells that lose WT1 (NB4, MV4-11, HEL, and primary AML cells) succumb to apoptosis (57-70% apoptosis after 24 h; p<0,001).2.The specific elimination of WT1 in K562 cells with morpholino antisense nucleotides (10 nM) significantly sensitizes the cells to hydroxyurea-induced apoptosis (26-30% increased apoptosis rates after 24 h; p<0,005).3.The hydroxyurea-induced loss of WT1 in sensitive cells is caspase-dependent but does not require the p53 tumor suppressor. The pan-caspase inhibitor ZVAD-FMK (20-50 µM) and the ectopic overexpression of mitochondrial anti-apoptotic proteins (BCL-XL/BCL2) prevents the hydroxyurea-induced depletion of WT1 and apoptosis in NB4 cells (from 62-69% to 9-14%; p<0,001).4.Moreover, our data reveal that hydroxyurea promotes the occurrence of previously unrecognized WT1 cleavage products. These are different from the known HTRA2-induced WT1 fragments, as they contain the WT1 zinc fingers required for DNA binding. These novel cleavage products also contain the domain for transcriptional activation, but lack the domain for transcriptional repression. We demonstrate that caspases cleave the full-length WT1 protein into such fragments, which accumulate in the nucleus of hydroxyurea-treated NB4 cells.5.Resistance of BCR-ABL-positive K562 CML cells to pro-apoptotic effects of hydroxyurea can be overcome with the tyrosine kinase inhibitor imatinib. This drug inhibits the WT1 promoter and sensitizes the cells to hydroxyurea (22-48% increased apoptosis rate after 24 h; p<0,005).6.The HDACi entinostat and panobinostat suppress the WT1 gene and accelerate the proteasomal degradation of WT1 by an increased expression of the E2 ubiquitin conjugase UBCH8. The depletion of WT1 by HDACi renders K562 cells sensitive towards hydroxyurea-induced killing and causes a synergistic activation of cell death (apoptosis increased by 43-55% after 24 h; p<0,001).7.In K562 cells hydroxyurea/entinostat combinations lead to a caspase-dependent cleavage of the DNA repair enzyme PARP-1 and to the accumulation of double-strand DNA breaks.8.We further show that entinostat significantly shifts the balance from a hydroxyurea-induced autophagy program to apoptosis (from 60% to 16% autophagy; p<0,001) in K562 cells. We reveal that entinostat depletes proteins critical for autophagy and autophagosome-lysosome fusion (Beclin-1 and HDAC6) in these cells.CONCLUSIONS:Our data illustrate that WT1 is a novel biological target of hydroxyurea, independent of a specific genetic lesion and irrespective of a functional p53 pathway. The inhibition of WT1 might be therapeutically exploitable for targeting approaches against leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

Analysis of WT1 Mutations and Combined Aberrations in Childhood Acute Myeloid Leukemia using Whole Exome Sequencing

Introduction:Mutations in the WT1 zinc finger gene (OMIM: 607102) are associated with an unfavorable clinical outcome in pediatric AML (Ho et al. 2010). In a subgroup of WT1mut patients additional mutations in other AML typical genes like FLT3 are also detectable. Balgobind et al. (2011) showed a worst outcome for pediatric patients with WT1-mutations combined with a FLT3-ITD (5-year overall survival=0,22, SE= 0,14). We and others identified combined mutations not only in the FLT3-TKD but also other genes like NPM1, nRAS or c-KIT. Using DNA from WT1mut AML pediatric patients with combined mutations, we performed whole exome sequencing (WES) to identify additional genetic aberrations to gain further insight into the genetic profile of this disease.Methods:From a cohort of 19 WT1mut patients, we selected 10 patients with CN and additional mutations in the FLT3-TKD, NPM1 or n-RAS gene. Concentration and quality of the purified genomic DNA (gDNA) was determined by Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). Fragmentation of 100ng gDNA was performed on a Covaris S2 (duty cycle 10%, intensity 4, 200 cycles per burst for 80s) to obtain fragments with an average length of 300 base pairs (bp). The DNA sequencing library was generated from 100ng of fragmented gDNA using Agilent SureSelectXT Reagent Kits v5 (50Mb) according to the manufacturer´s protocols. The final DNA sequencing library was purified, size-controlled by Agilent Technologies 2100 Bioanalyzer (High Sensitivity DNA Chip) and prepared for sequencing according to the manufacturer´s protocol (Illumina). The libraries were sequenced on Illumina HiSeq2500 using TruSeq SBS Kit v3-HS (200 cycles, paired end run) with an average of 12.5 x 106 reads per single exome (mean coverage: 50x). The GATK Pipeline (GenomeAnalysisTK-1.7) was applied for read quality trimming, read alignment to reference (hg19) and quality trimmed variant calling (McKenna et al., 2010). Variant annotation was done using Annovar (Wang et al., 2010).Results:In 19 out of 164 patients, who were screened for WT1, mutations in exon 7 or 9 of the WT1- gene were detected. Patients with WT1mut had an inferior 2-years event free survival compared to patients with WTwt (0.18, SE=0.09 vs. 0.61, SE=0.04, p=0,0001). DNA from WT1mut patients (n=10) with normal karyotype CN and additional mutations in the FLT3-TKD, NPM1- , or the n-RAS gene were elected for WES.The Annovar annotation suggests that each sample contains 5,359 – 5,480 genes that are mutated and where the mutations are predicted to have an effect. 2,324 of these mutated genes were present in all 10 samples together. After review of the literature, ZNF717, MUC16, MAP2K3, PRIM2, PKD1L2, MUC6, CDC27, TTN and MUC5B were identified as candidates for ongoing validation analysis. With respect to functional correlation, 90 - 113 frameshift deletions, 68 - 93 frameshift insertions, 103 - 125 non-frameshift deletions, and 96 - 118 non-frameshift insertions were predicted.Discussion:WT1mut AML pediatric patients with combined mutations in other AML- specific genes present a special subgroup with an adverse prognosis. Nevertheless, more information about the genetic network is necessary to understand the pathobiology and to improve treatment of the disease. For this purpose, we decided to perform whole exome sequencing in 10 samples from patients who were treated after the AML BFM 04 protocol. We identified mutations in more than 9 genes and several aberrations in functional regions of all samples. Currently, validation of these findings is being performed to evaluate the role of the mutated genes. Nevertheless, the results of this analysis will also shed also light onto the clonal evolution of the leukemic clone and the importance for the treatment and the prognosis of the patients. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2193: WT1 mediated regulation of growth control genes in leukemia

Abstract Prognosis and therapy of acute leukemia is influenced by leukemia-specific genetic alterations, thus highlighting the importance of identifying novel prognostic markers. One such potential marker, the Wilms’ tumor gene (WT1), a zinc finger transcription factor, is expressed in leukemic blasts and has been found to be mutated in approximately 10 percent of leukemia cases. Work in other tumor systems has shown that WT1 up-regulates expression of genes promoting cancer progression, including the angiogenic and mitogenic factor VEGF. Microarray studies have also correlated expression of JAG1 (Jagged 1) and CCNA1 (Cyclin A) with WT1 in Acute Myeloid Leukemia (AML) samples. We have identified potential WT1 binding sites within the promoters of both JAG1 and CCNA1 and have compared the expression of these genes to that of WT1, using real time quantitative PCR (QRTPCR), in pediatric leukemia and normal bone marrow. Compared to normal bone marrow, we observed lower than normal levels of WT1 in a majority of pediatric ALL samples, associated with lower than normal levels of VEGF, JAG-1 and CCNA-1. Conversely in pediatric AML(M3) we observed elevated levels of WT1 associated with elevated JAG-1 and CCNA-1. Mutations of the WT1 zinc finger (ZF) DNA binding domain have also been described in poor prognosis leukemias. To identify novel ZF mutations we sequenced WT1 in twelve pediatric acute leukemia samples. No ZF domain mutations were identified among these samples with high WT1 expression. However, a well-described SNP (rs 16754, also in exon 7), identified as a good prognostic marker in Cytogenetically Normal AML, was observed either as a homozygous or heterozygous variant of the WT1 gene. Because a majority of WT1 mutations identified in leukemias are frameshift mutations leading to a truncated protein lacking the ZF DNA binding domain, we created a truncation mutant of isoform A-WT1 lacking the ZF domain and assessed its function in K562 cells. Using QRTPCR we quantified the effect of over-expression of wild type and mutant WT1 on VEGF, JAG1 and CCNA1 expression. Compared to the vector control, wild type WT1 upregulated levels of JAG1 and CCNA1 but not VEGF mRNA. Mutant WT1 did not upregulate JAG1 or CCNA1. Our data and other studies have shown that JAG1 is elevated in AML patient samples, implicating the NOTCH 1 pathway that promotes cell proliferation and blocks differentiation. Thus, these results suggest that WT1 may act as an oncogene in K562 cells, in part by upregulating the growth promoting NOTCH-1 ligand, JAG1. Similarly, cell cycle regulation by WT1, demonstrated in other systems, may be mediated by transcriptional regulation of CCNA1. WT1 may contribute to leukemogenesis through transcriptional regulation of genes controlling cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2193. doi:1538-7445.AM2012-2193

Nuclear Transport of Wilms′ Tumour Protein Wt1 Involves Importins α and β

Background/Aims: Wilms′ tumour protein, Wt1, is a zinc finger molecule, which is required for normal embryonic development. Mutations of the WT1 gene can give rise to childhood cancer of the kidneys. Different Wt1 isoforms exist, which function either as transcription factors or have a presumed role in mRNA processing. Previous studies suggested that Wt1 undergoes nucleocytoplasmic shuttling, and cytoplasmic Wt1 was higher in malignant than in normal cells. The aim of this study was to analyse the molecular pathways along which Wt1 shuttles between the cytoplasm and nucleus. Methods: Interaction of Wt1 protein with various importin α subtypes and importin β was assessed in pull-down assays and co-immunoprecipitation experiments. Nuclear localisation signals (NLS) were identified by combining site-directed mutagenesis with subcellular immunodetection of the transfected Wt1 variants. Results: Wt1(+/-KTS) proteins were found to interact with importin α1 and importin β in vitro and in living cells in vivo. A NLS that was necessary and sufficient for nuclear import could be mapped to the third Wt1 zinc finger. Mutation of this NLS strongly weakened binding of Wt1 to importins. Conclusion: Nuclear translocation of Wilms′ tumour protein involves importins α and β, and a NLS in the third zinc finger.

Prevalence and prognostic implications of WT1 mutations in pediatric acute myeloid leukemia (AML): a report from the Children's Oncology Group

AbstractRecent studies of WT1 mutations in acute myeloid leukemia (AML) mostly report an association with unfavorable clinical outcome. We screened 842 patients treated on 3 consecutive pediatric AML trials for WT1 zinc-finger mutations. Eighty-five mutations were detected in 70 of 842 patients (8.3%). Mutations occurred predominantly in exon 7 (n = 74) but were also found in exons 8 (n = 5) and 9 (n = 6). Normal karyotype was observed in 35.3% of WT1mut patients, whereas 27.5% WT1mut patients harbored favorable risk cytogenetics. Patients with or without mutations had similar rates of complete remission after one course of induction chemotherapy. Overall survival (OS) for patients with WT1 mutations was 41% versus 54% for those without mutations (P = .016). Corresponding event-free survival (EFS) was also significantly worse for those with WT1 mutations (28% vs 42%; P = .01). However, FLT3/ITD was present in 36% of the WT1mut cohort; WT1mut patients without FLT3/ITD had similar OS (56% vs 56%, respectively; P = .8) and EFS (35% and 44%, respectively; P = .34) to patients who were wild type for both mutations. In current risk stratification schemes incorporating cytogenetics and FLT3/ITD status, the presence of WT1 mutations has no independent prognostic significance in predicting outcome in pediatric AML. The clinical trials are registered at www.clinicaltrials.gov as #NCT00002798 and #NCT00070174.

Contribution of Individual Amino Acids to the RNA Binding Activity of the Wilms’ Tumor Suppressor Protein WT1

In addition to binding to DNA, the zinc finger protein WT1 can also bind specifically to RNA. To determine the role of individual zinc fingers of the protein in this RNA binding activity, deletion and substitution mutants of the WT1 zinc finger domain were constructed. The effects of the various mutations on the binding of WT1 to the RNA aptamers RNA22 and RNA38 were determined using a quantitative equilibrium binding assay. The results indicate that zinc fingers 2 and 3 of WT1 are essential for the binding of the protein to the RNA aptamers. For both of these fingers, the arginine residue immediately preceding the alpha-helix makes a significant contribution to RNA binding. For zinc finger 2, a second arginine residue within the alpha-helix is also critical for RNA binding, while several alpha-helical residues in zinc finger 3 contribute to the overall affinity of WT1 for RNA. Investigating the effects of the same point mutations on DNA binding indicates that there are similarities and differences in the contributions of zinc fingers 2 and 3 to the DNA and RNA binding activities of WT1.

Structure of the Wilms Tumor Suppressor Protein Zinc Finger Domain Bound to DNA

The zinc finger domain of the Wilms tumor suppressor protein (WT1) contains four canonical Cys 2His 2 zinc fingers. WT1 binds preferentially to DNA sequences that are closely related to the EGR-1 consensus site. We report the structure determination by both X-ray crystallography and NMR spectroscopy of the WT1 zinc finger domain in complex with DNA. The X-ray structure was determined for the complex with a cognate 14 base-pair oligonucleotide, and composite X-ray/NMR structures were determined for complexes with both the 14 base-pair and an extended 17 base-pair DNA. This combined approach allowed unambiguous determination of the position of the first zinc finger, which is influenced by lattice contacts in the crystal structure. The crystal structure shows the second, third and fourth zinc finger domains inserted deep into the major groove of the DNA where they make base-specific interactions. The DNA duplex is distorted in the vicinity of the first zinc finger, with a cytidine twisted and tilted out of the base stack to pack against finger 1 and the tip of finger 2. By contrast, the composite X-ray/NMR structures show that finger 1 continues to follow the major groove in the solution complexes. However, the orientation of the helix is non-canonical, and the fingertip and the N terminus of the helix project out of the major groove; as a consequence, the zinc finger side-chains that are commonly involved in base recognition make no contact with the DNA. We conclude that finger 1 helps to anchor WT1 to the DNA by amplifying the binding affinity although it does not contribute significantly to binding specificity. The structures provide molecular level insights into the potential consequences of mutations in zinc fingers 2 and 3 that are associated with Denys-Drash syndrome and nephritic syndrome. The mutations are of two types, and either destabilize the zinc finger structure or replace key base contact residues.

Hormone treatment enhances WT1 activation of Renilla luciferase constructs in LNCaP cells

The zinc finger transcription factor, WT1, regulates many growth control genes, repressing or activating transcription depending on the gene and cell type. Based on earlier analyses of the effect of WT1 on androgen responsive genes, we hypothesized that there may be an interaction between the androgen signaling pathway and WT1, such that the commonly used Renilla luciferase control vectors were activated in LNCaP prostate cancer cells. Using cotransfection assays we tested the effects of WT1 and/or the androgen analog, R1881, on two Renilla luciferase vectors, pRL-SV40 and the promoter-less pRL-null. To determine whether the zinc finger DNA binding domain was required, the zinc finger mutant DDS-WT1 (R394W) was tested; but it had no significant effect on the Renilla luciferase vectors. To determine whether the androgen signaling pathway was required, WT1 was co-transfected with Renilla vectors in cells with varied hormone responsiveness. The WT1 effect on pRL-null varied from no significant effect in 293 and PC3 cells to very strong enhancement in LNCaP cells treated with 5 nM R1881. Overall, these results suggest that hormone enhanced WT1 mediated activation of Renilla luciferase and that these interactions require an intact WT1 zinc finger DNA binding domain.

The Wilms tumour suppressor protein WT1 (+KTS isoform) binds alpha-actinin 1 mRNA via its zinc-finger domain

Mutations in WT1 are associated with developmental syndromes that affect the urogenital system and neoplasms, including Wilms tumour, acute myeloid leukemia, and breast and prostate cancers. The WT1 protein belongs to the early growth response family of zinc-finger transcription factors. Uniquely to WT1, an evolutionarily conserved alternative splice event inserts the tripeptide KTS, between zinc fingers 3 and 4. Whereas -KTS isoforms bind DNA and activate or repress transcription, +KTS isoforms bind DNA less efficiently and interact with splice factors and RNA in vitro and in vivo. Although candidate DNA targets have been found, physiological mRNA targets are yet to be defined. We examined the distribution of WT1 in ribonucleoprotein (RNP) complexes in nuclear extract prepared from M15 cells, a mouse mesonephric fetal kidney cell line. WT1 cofractionated with the splice factor PSF in large RNP particles >or=2 MDa. We also found that PSF co-immunoprecipitated with WT1, suggesting a functional interaction between these 2 multifunctional proteins. Using yeast three-hybrid library constructed from the co-immunoprecipitated RNA we found that WT1 (+KTS) binds close to or at the start codon of alpha-actinin 1 (ACTN1) mRNA. A band shift assay confirmed the ability of the WT1 zinc-finger domain (+KTS) to bind this sequence in vitro. ACTN1 is the first likely physiological mRNA target of WT1.

HnRNP-U directly interacts with WT1 and modulates WT1 transcriptional activation

The Wilms' tumour suppressor gene, WT1, encodes a zinc-finger protein that is mutated in Wilms' tumours and highly expressed in a wide variety of other malignancies. WT1 is a transcription factor that is likely to have additional, post-transcriptional, regulatory roles, although the molecular mechanisms by which WT1 acts remain poorly understood. We have combined genetic and biochemical approaches to show, that endogenous WT1 binds to heterogeneous nuclear ribonuclear protein U (hnRNP-U), that this interaction does not require any other proteins or nucleic acids, involves the zinc-fingers of WT1 and the middle domain of hnRNP-U, and that hnRNP-U can modulate WT1 transcriptional activation of a bona fide WT1 target gene. These findings increase our knowledge of how WT1 exerts its transcriptional regulatory role and suggests that hnRNP-U may be a candidate Wilms' tumour gene at 1q44.

Upregulation of Wilms' tumor gene 1 (WT1) in desmoid tumors

Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors in which beta-catenin/TCF3 mediated Wnt signaling is activated. More than 80% of desmoid tumors contain activating mutations in beta-catenin. It has been shown that the Wnt signaling pathway interacts with Wilms' tumor gene 1 (WT1) in normal kidney development and plays a role in the genesis of some Wilms' tumors. About 15% of Wilms' tumors contain WT1 mutations and of these, about 50% contain beta-catenin mutations. This overlap in mutation pattern of WT1 and beta-catenin in Wilms' tumor suggests that these 2 genes may collaborate in the genesis of a subset of Wilms' tumors. To investigate whether this hypothesis could be extended to other Wnt-dependent tumor types, we searched for WT1 mutations and studied WT1 expression in beta-catenin mutant desmoid tumors. We investigated the expression of WT1 mRNA and protein in desmoid tumors. Medium to high abundant levels of WT1 mRNA were detected by TaqMan quantitative PCR in all tested desmoid cells, whereas adjacent normal fibroblasts showed less expression of WT1. Western blot analysis and immunohistochemistry confirmed this overexpression at the protein level. A mutational screen of the WT1 zinc-finger region by sequence analysis did not identify any mutations. Finally, we investigated a possible role of beta-catenin on WT1 regulation and vice versa. Overexpression of different beta-catenin mutants in the HEK293T cell line did not modulate WT1 promoter activity and WT1 did not affect beta-catenin /TCF transcriptional activity in this cell line. These results show that the wild-type WT1 gene is strongly overexpressed in beta-catenin mutant desmoid tumors and may play a role in tumorigenesis of desmoid tumors, similar to what has been suggested in some epithelial malignancies.

Induction of the interleukin-2/15 receptor beta-chain by the EWS-WT1 translocation product.

EWS-WT1 is a chimeric transcription factor resulting from fusion of the N-terminal domain of the Ewing sarcoma gene EWS to the three C-terminal zinc fingers of the Wilms tumor suppressor WT1. This translocation underlies desmoplastic small round cell tumor (DSRCT), which is noted for the abundance of reactive stroma surrounding islets of tumor cells, suggestive of paracrine signals contributing to tumor cell proliferation. Hybridization to high-density oligonucleotide microarrays can be used to identify targets of EWS-WT1. Expression of EWS-WT1 from a tetracycline-regulated promoter leads to the induction of growth-associated genes, of which the most remarkable is the beta-chain of the interleukin-2/15 receptor (IL-2/15Rbeta). Potent transcriptional activation by the chimeric protein maps to two bindings sites within the IL-2/15Rbeta promoter. Analysis of primary DSRCT tumor specimens demonstrates high levels of IL-2/15Rbeta within the tumor cells, along with expression of IL-2 and IL-15 by the abundant hyperplastic endothelial cells within the reactive stroma. Activation of this cytokine signaling pathway is consistent with the nuclear localization of its downstream effectors, phosphorylated STAT3 and STAT5. These observations suggest that the transcriptional induction of a cytokine receptor by a tumor-associated translocation product enables a proliferative response of epithelial cancer cells to ligands secreted by the surrounding stroma.

A Functional Interaction with CBP Contributes to Transcriptional Activation by the Wilms Tumor Suppressor WT1

The Wilms tumor gene WT1 encodes a zinc finger transcription factor that is required for normal kidney development. WT1 was identified as a transcriptional repressor, based on its suppression of promoter reporters, but analysis of native transcripts using high density microarrays has uncovered transcriptional activation, rather than repression, of potential target genes. We report here that WT1 binds to the transcriptional coactivator CBP, leading to synergistic activation of a physiologically relevant promoter. The physical interaction between WT1 and CBP is evident in vitro and in vivo, and the two proteins are co-immunoprecipitated from embryonic rat kidney cells. The WT1-CBP association requires the first two zinc fingers of WT1 and the adenovirus 5 E1A-binding domain of CBP. Overexpression of this domain of CBP is sufficient to inhibit WT1-mediated transcriptional activation of a promoter reporter, as is co-transfection of E1A. Retrovirally driven expression of either the CBP fragment or of E1A in human hematopoietic cells suppresses the induction by WT1 of its endogenous target gene, p21(Cip1). These observations support a model of WT1 as a transcriptional activator of genes required for cellular differentiation.

Wilms Tumor and the WT1 Gene

Wilms tumor or nephroblastoma is a pediatric kidney cancer arising from pluripotent embryonic renal precursors. Multiple genetic loci have been linked to Wilms tumorigenesis; positional cloning strategies have led to the identification of the WT1 tumor suppressor gene at chromosome 11p13. WT1 encodes a zinc finger transcription factor that is inactivated in the germline of children with genetic predisposition to Wilms tumor and in a subset of sporadic cancers. When present in the germline, specific heterozygous dominant-negative mutations are associated with severe abnormalities of renal and sexual differentiation, pointing to the essential role of WT1 for normal genitourinary development. The role of this tumor suppressor in normal organ-specific differentiation is also supported by the highly restricted temporal and spatial expression of WT1 in glomerular precursors of the developing kidney and by the failure of kidney development in wt1-null mice. Of two major alternative splicing products encoded by WT1, the (−KTS) isoform appears to mediate transcriptional activation of genes implicated in cellular differentiation, possibly also repressing proliferation-associated genes. The (+KTS) isoform, whose DNA-binding domain is disrupted by the insertion of three amino acids, may be involved in some aspect of mRNA processing. In addition to its function in genitourinary development, a role for WT1 in hematopoiesis is suggested by its aberrant expression and/or mutation in a subset of acute human leukemias. WT1 is also expressed in mesothelial cells; a specific oncogenic chromosomal translocation fusing the N-terminal domain of the Ewing sarcoma gene EWS to the three C-terminal zinc fingers of WT1 underlies desmoplastic small round cell tumor, an abdominal tumor thought to arise from the peritoneal lining. Understanding the distinct functional properties of WT1 isoforms and tumor-associated variants will provide unique insight into the link between normal organ-specific differentiation and malignancy.

E1B 55K sequesters WT1 along with p53 within a cytoplasmic body in adenovirus-transformed kidney cells.

WT1 encodes a tumor suppressor that is expressed in cells of the developing kidney and is inactivated in Wilms tumor, a pediatric kidney cancer. The adenovirus E1B 55K gene product contributes to the transformation of primary baby rat kidney (BRK) cells by binding and inactivating the product of the p53 tumor suppressor. We have previously demonstrated that WT1 and p53 are present within a protein complex in vivo. We now show that WT1 is physically associated with E1B 55K in adenovirus-transformed cells, an interaction that is mediated by the first two zinc fingers of WT1. Immunodepletion of p53 abrogates the coimmunoprecipitation of E1B 55K and WT1, consistent with the presence of a trimeric protein complex containing these three proteins. In the presence of E1B 55K, WT1 which is normally localized in the nucleus, is retained within a very high molecular weight complex and sequestered in the characteristic perinuclear cytoplasmic body that contains E1B 55K and p53. Expression of E1B 55K in osteosarcoma cells that undergo apoptosis following expression of WT1 inhibits WT1-mediated cell death. We conclude that E1B 55K may target WT1 along with p53, resulting in the functional inactivation of both tumor suppressor gene products by this viral oncoprotein.

The desmoplastic small round cell tumor t(11;22) translocation produces EWS/WT1 isoforms with differing oncogenic properties.

Structural alterations of the Wilms' tumor locus (WT1) at 11p13 have been implicated in the etiology of two human cancers--Wilms' tumor (WT), a pediatric renal malignancy, and Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer of the abdominal serosal lining with predilection for male adolescents. Germline mutations within the WT1 tumor suppressor gene predispose to WT and are associated with congenital malformations of the urogenital system, and somatic mutations are associated with initiation of transformation in WTs. In DSRCT, a recurrent translocation, t(11;22)(p13;q12), fuses the amino terminal domain of the EWS1 gene product to three of the four WT1 zinc fingers. Two EWS/WT1 isoforms are generated as a result of an alternative splicing event between zinc fingers III and IV, inserting or removing three amino acids (+/- KTS). We demonstrate that introduction of EWS/WT1(-KTS) into NIH3T3 cells causes their tumorigenic transformation as determined by: formation of transformed foci on a monolayer of cells; anchorage-independent growth; and tumor formation in nude mice. EWS/WT1(+KTS) showed no transforming potential in these assays. These results indicate the oncogenic effect of the t(11;22) translocation is mediated by the EWS/WT1(-KTS) isoform and that fusion of the EWS amino terminal domain to the WT1 DNA binding domain produces a chimeric product showing a gain of function.

The DNA binding domains of the WT1 tumor suppressor gene product and chimeric EWS/WT1 oncoprotein are functionally distinct.

The t(11;22)(p13;q12) translocation associated with desmosplastic small round cell tumor results in a chimeric molecule fusing the amino terminal domain (NTD) of the EWS1 gene to three of the four carboxy-terminal zinc fingers of the WT1 tumor suppressor gene. Since the DNA binding domains of WT1 and EWS/WT1 are structurally different, we have assessed the functional consequences of the EWS/WT1 fusion. We find that the EWS/WT1 protein has a higher binding affinity for a given recognition target than the WT1 product. This is unlike other fusion products involving translocation of the NTD of EWS to DNA binding domains in which DNA binding specificity and affinity is not changed. We demonstrate that EWS/WT1 is a nuclear protein and that the NTD of EWS contains (a) nuclear localization signal(s). We also find that the integrity of a domain within the WT1 zinc fingers, responsible for mediating interaction between WT1 and the transcriptional repressor par-4, is disrupted in the EWS/WT1 fusion product. Deletion analysis of the NTD of EWS indicated that integrity of the entire domain was necessary to achieve full transactivation potential.