Christopher T. Denny

The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1.

EWS/FLI-1 is a chimeric protein formed by a tumor-specific 11;22 translocation found in both Ewings sarcoma and primitive neuroectodermal tumor of childhood. EWS/FLI-1 has been shown to be a potent transforming gene, suggesting that it plays an important role in the genesis of these human tumors. We now demonstrate that EWS/FLI-1 has the characteristics of an aberrant transcription factor. Subcellular fractionation experiments localized the EWS/FLI-1 protein to the nucleus of primitive neuroectodermal tumor cells. EWS/FLI-1 specifically bound in vitro an ets-2 consensus sequence similarly to normal FLI-1. When coupled to a GAL4 DNA-binding domain, the amino-terminal EWS/FLI-1 region was a much more potent transcriptional activator than the corresponding amino-terminal domain of FLI-1. Finally, EWS/FLI-1 efficiently transformed NIH 3T3 cells, but FLI-1 did not. These data suggest that EWS/FLI-1, functioning as a transcription factor, leads to a phenotype dramatically different from that of cells expressing FLI-1. EWS/FLI-1 could disrupt normal growth and differentiation either by more efficiently activating FLI-1 target genes or by inappropriately modulating genes normally not responsive to FLI-1.

Leukemia and the disruption of normal hematopoiesis

The goal of this review is to highlight recent advances in our understanding of the pathobiology of leukemias and lymphomas. Our theme throughout will be to relate specific mechanisms involved in the initiation and progression of leukemia to the alteration of the normal homeostatic mechanisms used to regulate the production of blood cells. Advances in the definition of hematopoietic growth factors and their receptors and cellular oncogenes provide a reservoir of possibilities to consider for growth deregulation. When possible we will concentrate on mechanisms defined for human leukemias and lymphomas and use data from experimental animal models for contrast or to demonstrate unique concepts. Although many of the points we will describe could relate to most leukemias (e.g., clonality, or block to differentiation), we have chosen specific leukemias or lymphomas to exemplify such concepts. Advancement in the isolation and characterization of the pluripotent stem cell has furthered our understanding of its related malignancies. Most leukemias, however, have classically been categorized as lymphoid or myeloid (Figure 1). We have favored grouping hematopoietic neoplasias according to related molecular and cell biologic mechanisms. We will discuss selected examples that appear to function as regulators of cell growth or differentiation. We define leukemia as the uncontrolled proliferation

Biology of EWS/ETS fusions in Ewing's family tumors

Tumor-associated chromosomal translocations lead to the formation of chimeric fusions between the EWS gene and one of five different ETS transcription factors in Ewings family tumors (EFTs). The resultant EWS/ETS proteins promote oncogenesis in a dominant fashion in model systems and are necessary for continued growth of EFT cell lines. EWS belongs to a family of genes that encode proteins that may serve as adapters between the RNA polymerase II complex and RNA splicing factors. EWS/ETS fusions have biochemical characteristics of aberrant transcription factors and appear to promote abnormal cellular growth by transcriptionally modulating a network of target genes. Early evidence suggests that EWS/ETS proteins may also impact gene expression through alteration in RNA processing. Elucidation of EWS/ETS target gene networks in the context of other signaling pathways will hopefully lead to biology based therapeutic strategies for EFT.

Identification of target genes for the Ewing's sarcoma EWS/FLI fusion protein by representational difference analysis.

The EWS/FLI-1 fusion gene results from the 11;22 chromosomal translocation in Ewings sarcoma. The product of the gene is one of a growing number of structurally altered transcription factors implicated in oncogenesis. We have employed a subtractive cloning strategy of representational difference analysis in conjunction with a model transformation system to identify genes transcribed in response to EWS/FLI. We have characterized eight transcripts that are dependent on EWS/FLI for expression and two transcripts that are repressed in response to EWS/FLI. Three of the former were identified by sequence analysis as stromelysin 1, a murine homolog of cytochrome P-450 F1 and cytokeratin 15. Stromelysin 1 is induced rapidly after expression of EWS/FLI, suggesting that the stromelysin 1 gene may be a direct target gene of EWS/FLI. These results demonstrate that expression of EWS/FLI leads to significant changes in the transcription of specific genes and that these effects are at least partially distinct from those caused by expression of germ line FLI-1. The representational difference analysis technique can potentially be applied to investigate transformation pathways activated by a broad array of genes in different tumor systems.

Structural basis for the interaction of the free SH2 domain EAT-2 with SLAM receptors in hematopoietic cells

The T and natural killer (NK) cell‐specific gene SAP (SH2D1A) encodes a ‘free SH2 domain’ that binds a specific tyrosine motif in the cytoplasmic tail of SLAM (CD150) and related cell surface proteins. Mutations in SH2D1A cause the X‐linked lymphoproliferative disease, a primary immunodeficiency. Here we report that a second gene encoding a free SH2 domain, EAT‐2, is expressed in macrophages and B lympho cytes. The EAT‐2 structure in complex with a phosphotyrosine peptide containing a sequence motif with Tyr281 of the cytoplasmic tail of CD150 is very similar to the structure of SH2D1A complexed with the same peptide. This explains the high affinity of EAT‐2 for the pTyr motif in the cytoplasmic tail of CD150 but, unlike SH2D1A, EAT‐2 does not bind to non‐phosphorylated CD150. EAT‐2 binds to the phosphorylated receptors CD84, CD150, CD229 and CD244, and acts as a natural inhibitor, which interferes with the recruitment of the tyrosine phosphatase SHP‐2. We conclude that EAT‐2 plays a role in controlling signal transduction through at least four receptors expressed on the surface of professional antigen‐presenting cells.

Divergent Ewing's sarcoma EWS/ETS fusions confer a common tumorigenic phenotype on NIH3T3 cells

Ewings sarcomas express chimeric transcription factors resulting from a fusion of the amino terminus of the EWS gene to the carboxyl terminus of one of five ETS proteins. While the majority of tumors express EWS/FLI1 fusions, some Ewings tumors contain variant chimeras such as EWS/ETV1 that have divergent ETS DNA-binding domains. In spite of their structural differences, both EWS/ETS fusions up regulate EAT-2, a previously described EWS/FLI1 target gene. In contrast to EWS/FLI1, NIH3T3 cells expressing EWS/ETV1 cannot form colonies in soft agar though coexpression of a dominant negative truncated ETV1 construct attenuates EWS/FLI1 mediated anchorage independent growth. When EWS/ETV1 or EWS/FLI1 expressing NIH3T3 cells are injected into SCID mice, tumors form more often and faster than with NIH-3T3 cells with empty vector controls. The tumorigenic potency of each EWS/ETS fusion is linked to its C-terminal structure, with the FLI1 C-terminus confering a greater tumorigenic potential than the corresponding ETV1 domain. The resulting EWS/ETV1 and EWS/FLI1 tumors closely resemble each other at both a macroscopic and a microscopic level. These tumors differ greatly from tumors formed by NIH3T3 cells expressing activated RAS. These data indicate that in spite of their structural differences, EWS/ETV1 and EWS/FLI1 promote oncogenesis via similar biologic pathways.

Loss of p16 pathways stabilizes EWS/FLI1 expression and complements EWS/FLI1 mediated transformation.

Ewings sarcoma and primitive neuroectodermal tumors (ES/PNET) are characterized by the fusion of the N-terminus of the EWS gene to the C-terminus of a member of the ETS family of transcription factors. While such fusion proteins are thought to play dominant oncogenic roles, it is unlikely that a single genetic alteration by itself will support cellular transformation. Given that EWS/FLI1 is only able to transform immortalized 3T3 fibroblasts and that 30% of ES/PNET tumors contain a homozygous deletion of the p16 locus, it is likely that other genetic events are required for EWS/FLI1 oncogenesis. Here we describe a complementary mechanism utilized in the establishment ES/PNET tumors. EWS/FLI1 has the capacity to induce apoptosis and growth arrest in normal MEFs. Such effects prevent the establishment of stable expression of the protein in these cells. When expressed in p16, p19ARF, or p53 deficient MEFs, the apoptotic and growth arrest effects are attenuated, creating a environment permissive for stable expression of the protein. While loss of a single tumor suppressor is sufficient to establish expression of EWS/FLI1, cellular transformation requires further genetic perturbation.

Chronic myelomonocytic leukemia: Tel-a-kinase what Ets all about.

Department of Medicine tDepartment of Pediatrics Divisions of Hematology-Oncology Gwynne Hazen Memorial Laboratory Molecular Biology Institute and Jonsson Comprehensive Cancer Center University of California, Los Angeles Los Angeles, California 90024 A logical approach toward identifying the genes responsi- ble for cancer is to clone tumor-specific chromosomal ab- normalities such as translocation breakpoints. For human leukemias, transcription factors are at the heart of many breakpoints. In this issue of Cell, Golub et al. (1994) report the cloning of the t(5;12) translocation found in patients with chronic myelomonocytic leukemia (CMML). Although their approach is a familiar one, the result is quite surpris- ing. This translocation fuses a member of the Ets family of transcription factors named Tel (for translocation, Ets, leukemia) to a well-known receptor tyrosine kinase (RTK), the platelet-derived growth factor receptor j3 (PDGFRP). A kinase-transcription factor fusion raises novel questions about how such chimeric genes might function. Further- more, the discovery that an altered PDGFRB protein plays a role in CMML adds to the accumulating ring of evidence that Ras abnormalities are central to the genesis of multi- ple types of myeloid leukemia. RTK Fusion Oncogenes: Constifutive Dimerization Activates Normal Signal Transduction Pathways A first step in understanding how the Tel-PDGFRP fusion protein might lead to leukemia is to examine the range of previously described translocations involving tyrosine kinases. We have limited our comparison to transmem- brane RTKs to keep the analogy to PDGFRp as close as possible. Figure 1 summarizesselected examplesof RTKs converted to oncogenes by slightly different structural al- terations. We must know how normal tyrosine kinase receptors function to understand how mutant receptors might work. A critical step initiated by ligand binding is dimerization of two adjacent receptors followed by phosphorylation of specific tyrosine residues in the cytoplasmic tails of the paired receptors. These phosphorylated tyrosine residues are high affinity-binding sites for the Src homology 2 (SH2) domains of specific cytoplasmic signaling molecules such as GrbP. The SH2 domains of these molecules dock onto the tail of the dimerized receptor, forming a complex which transmits the signal into the cell. In the case of GrbP, the consequence is clear (reviewed by Schlessinger, 1993). In addition to binding the receptor, Grb2 binds the GDP- GTP exchanger molecule Son of sevenless (SOS), which, in turn, activates the Ras protein (Figure 2). The most straightforward explanation for how mutant versions of these tyrosine kinase receptors function as oncogenes is that they represent constitutively activated

EWS/FLI1 up regulates mE2-C , a cyclin-selective ubiquitin conjugating enzyme involved in cyclin B destruction

The EWS/FLI1 fusion gene found in Ewings sarcoma and primitive neuroectodermal tumor, is able to transform certain cell lines by acting as an aberrant transcription factor. The ability of EWS/FLI1 to modulate gene expression in cells transformed and resistant to transformation by EWS/FLI1, was assessed by Representational Difference Analysis (RDA). We found that the cyclin selective ubiquitin conjugase murine E2-C, was up regulated in NIH3T3 cells transformed by EWS/FLI1 but not in a nontransformed NIH3T3 clone expressing EWS/FLI1. We also found that mE2-C is upregulated in NIH3T3 cells transformed by other genes including activated cdc42, v-ABL and c-myc. We demonstrated that expression of mE2-C in both the EWS/FLI1 transformed and parent NIH3T3 lines varies with the cell cycle. Finally, dominant-negative mE2-C, created by changing a catalytic cysteine to serine, inhibits the in vitro ubiquitination and degradation of cyclin B in human HeLa cell extracts. These data suggest that part of the biologic effect of EWS/FLI1 could be to transcriptionally modulate genes involved in cell cycle regulation.

Multiple aromatic side chains within a disordered structure are critical for transcription and transforming activity of EWS family oncoproteins.

Chromosomal translocations involving the N-terminal ≈250 residues of the Ewings sarcoma (EWS) oncogene produce a group of EWS fusion proteins (EFPs) that cause several distinct human cancers. EFPs are potent transcriptional activators and interact with other proteins required for mRNA biogenesis, indicating that EFPs induce tumorigenesis by perturbing gene expression. Although EFPs were discovered more than a decade ago, molecular analysis has been greatly hindered by the repetitive EWS activation domain (EAD) structure, containing multiple degenerate hexapeptide repeats (consensus SYGQQS) with a conserved tyrosine residue. By exploiting total gene synthesis, we have been able to systematically mutagenize the EAD and determine the effect on transcriptional activation by EWS/ATF1 and cellular transformation by EWS/Fli1. In both assays, we find the following requirements for EAD function. First, multiple tyrosine residues are essential. Second, phenylalanine can effectively substitute for tyrosine, showing that an aromatic ring can confer EAD function in the absence of tyrosine phosphorylation. Third, there is little requirement for specific peptide sequences and, thus, overall sequence composition (and not the degenerate hexapeptide repeat) confers EAD activity. Consistent with the above findings, we also report that the EAD is intrinsically disordered. However, a sensitive computational predictor of natural protein disorder (PONDR VL3) identifies potential molecular recognition features that are tyrosine-dependent and that correlate well with EAD function. In summary we have uncovered several molecular features of the EAD that will impact future studies of the broader EFP family and molecular recognition by complex intrinsically disordered proteins.