The target cell of transformation is distinct from the leukemia stem cell in murine CALM/AF10 leukemia models.

The CALM/AF10 fusion gene is found in various hematological malignancies including acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia and malignant lymphoma. We have previously identified the leukemia stem cell (LSC) in a CALM/AF10-driven murine bone marrow transplant AML model as B220+ lymphoid cells with B-cell characteristics. To identify the target cell for leukemic transformation or 'cell of origin of leukemia' (COL) in non-disturbed steady-state hematopoiesis, we inserted the CALM/AF10 fusion gene preceded by a loxP-flanked transcriptional stop cassette into the Rosa26 locus. Vav-Cre-induced panhematopoietic expression of the CALM/AF10 fusion gene led to acute leukemia with a median latency of 12 months. Mice expressing CALM/AF10 in the B-lymphoid compartment using Mb1-Cre or CD19-Cre inducer lines did not develop leukemia. Leukemias had a predominantly myeloid phenotype but showed coexpression of the B-cell marker B220, and had clonal B-cell receptor rearrangements. Using whole-exome sequencing, we identified an average of two to three additional mutations per leukemia, including activating mutations in known oncogenes such as FLT3 and PTPN11. Our results show that the COL for CALM/AF10 leukemia is a stem or early progenitor cell and not a cell of B-cell lineage with a phenotype similar to that of the LSC in CALM/AF10+ leukemia.

High-level expression of Mastermind-like 2 contributes to aberrant activation of the NOTCH signaling pathway in human lymphomas.

Inappropriate activation of the NOTCH signaling pathway, for example, by activating mutations, contributes to the pathogenesis of various human malignancies. Here, we demonstrate that aberrant expression of an essential NOTCH coactivator of the Mastermind-like (MAML) family provides an alternative mechanism to activate NOTCH signaling in human lymphoma cells. We detected high-level MAML2 expression in several B cell-derived lymphoma types, including classical Hodgkin lymphoma (cHL) cells, relative to normal B cells. Inhibition of MAML-protein activity by a dominant negative form of MAML or by small hairpin RNAs targeting MAML2 in cHL cells resulted in downregulation of the NOTCH target genes HES7 and HEY1, which we identified as overexpressed in cHL cells, and in reduced proliferation. Furthermore, a NOTCH gene-expression signature in cHL cells confirmed their cell-autonomous NOTCH activity. Finally, in line with the essential role of MAML proteins for assembly and activity of the NOTCH transcriptional complex (NTC), we show that MAML-derived small-peptide constructs block NOTCH activity and disrupt NTC formation in vitro. These data strongly suggest direct targeting of the NTC as treatment strategy for NOTCH-dependent malignancies.

EBNA2 and Notch signalling in Epstein-Barr virus mediated immortalization of B lymphocytes.

Epstein-Barr virus (EBV) has the ability to immortalize B cells. A viral key protein for immortalization is the transactivator EBNA2 that controls expression of several viral and cellular genes. EBNA2 is tethered to promoters by interacting with the cellular repressor RBP-J. This resembles the physiological activation of RBP-J-repressed promoters by activated Notch receptors (Notch-IC). Since EBNA2 and Notch-IC have been shown to be partially interchangeable in regard to activation of target genes in B cell lines and modulation of differentiation processes it is conceivable that EBNA2 is a biological equivalent of an activated Notch receptor.

Activated Notch1 can transiently substitute for EBNA2 in the maintenance of proliferation of LMP1-expressing immortalized B cells.

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for immortalization of human B cells by EBV. EBNA2 and activated Notch transactivate genes by interacting with the cellular transcription factor RBP-Jkappa/CBF1. Therefore, EBNA2 can be regarded as a functional homologue of activated Notch. We have shown previously that the intracellular domain of Notch1 (Notch1-IC) is able to transactivate EBNA2-regulated viral promoters and to induce phenotypic changes in B cells similar to those caused by EBNA2. Here we investigated whether Notch1-IC can substitute for EBNA2 in the maintenance of B-cell proliferation. Using an EBV-immortalized lymphoblastoid cell line in which EBNA2 function can be regulated by estrogen, we demonstrate that murine Notch1-IC, in the absence of functional EBNA2, is unable to maintain LMP1 expression and to maintain cell proliferation. However, in a lymphoblastoid cell line expressing LMP1 independently of EBNA2, murine Notch1-IC can transiently maintain proliferation after EBNA2 inactivation. After 4 days, cell numbers do not increase further, and cells in the G2 phase of the cell cycle start to die. In contrast to EBNA2, murine Notch1-IC is unable to upregulate the expression of the c-myc gene in these cells.

Activated Notch1 modulates gene expression in B cells similarly to Epstein-Barr viral nuclear antigen 2.

Both Epstein-Barr viral nuclear antigen 2 (EBNA2) and activated Notch transactivate genes by interacting with the transcription factor RBP-Jkappa. The viral protein EBNA2 may hence be regarded as a functional equivalent of an activated Notch receptor. Until now, nothing has been known about the physiological role of Notch signaling in B cells. Here we investigated whether activated Notch can induce the same phenotypic changes as EBNA2 in Burkitt's lymphoma cells. An estrogen receptor fusion protein of the intracellular part of mouse Notch 1 (mNotch1-IC), mimicking in the presence of estrogen a constitutively active Notch receptor, was stably transfected into the Burkitt's lymphoma cell lines BL41-P3HR1 and HH514. Northern blot analysis revealed that the LMP2A gene is induced by Notch-IC in the presence of estrogen, whereas increased expression of LMP1 could be detected only if cycloheximide was simultaneously added. Concerning the cellular genes regulated by EBNA2, Notch-IC was able to upregulate CD21 but not CD23 expression. Immunoglobulin mu (Igmu) expression, which is downregulated by EBNA2, was also negatively regulated by Notch-IC. Similarly to EBNA2, Notch-IC was able to repress c-myc expression, which is under the control of the immunoglobulin heavy-chain locus in Burkitt's lymphoma cells with a t(8;14) translocation. The data show that Notch-IC is able to participate in gene regulation in B cells.

Activated mouse Notch1 transactivates Epstein-Barr virus nuclear antigen 2-regulated viral promoters.

Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for B-cell immortalization by EBV, most probably by its ability to transactivate a number of cellular and viral genes. EBNA2-responsive elements (EBNA2REs) have been identified in several EBNA2-regulated viral promoters, each of them carrying at least one RBP-Jkappa recognition site. RBP-Jkappa recruits EBNA2 to the EBNA2RE and, once complexed to EBNA2, is converted from a repressor into an activator. An activated form of the cellular receptor Notch also interacts with RBP-Jkappa, providing a link between EBNA2 and Notch signalling. To determine whether activated Notch is able to transactivate EBNA2-responsive viral promoters, we performed cotransfection experiments with activated mouse Notch1 (mNotch1-IC) and luciferase constructs of the BamHI C, LMP1, and LMP2A promoters. We present here evidence that mNotch1-IC transactivates viral promoters known to be regulated by EBNA2. As shown for EBNA2, mutations or deletions of the RBP-Jkappa sites diminish or eliminate mNotch1-IC-mediated transactivation of the promoters, pointing to an essential role for Notch-RBP-Jkappa interaction. In addition to RBP-Jkappa, other cellular factors may bind within the EBNA2REs of viral promoters. While some factors appear to play an important role in both EBNA2- and mNotch1-IC-mediated transactivation, others are only important for the activity of either EBNA2 or mNotch1-IC. We could observe specific mNotch1-IC-responsive regions, thereby throwing more light upon which cofactors interact with EBNA2 and mNotch1-IC, thus enabling them to become functionally transactivators in vivo.

Functional replacement of the intracellular region of the Notch1 receptor by Epstein-Barr virus nuclear antigen 2.

The intracellular region (RAMIC) of the mouse Notch1 receptor interacts with RBP-J/CBF-1, which binds to the DNA sequence CGTGGGAA and suppresses differentiation by transcriptional activation of genes regulated by RBP-J. Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for immortalization of human B cells by the virus. EBNA2 is a pleiotropic activator of viral and cellular genes and is targeted to DNA at least in part by interacting with RBP-J. We found that EBNA2 and the Notch1 RAMIC compete for binding to RBP-J, indicating that their interaction sites on RBP-J overlap at least partially. EBNA2 and Notch1 RAMIC transactivated the same set of viral and host promoters, i.e., the EBNA2 response element of the Epstein-Barr virus TP1 and the HES-1 promoter. Furthermore, EBNA2 functionally replaced the Notch1 RAMIC by suppressing differentiation of C2C12 myoblast progenitor cells.

Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.

RBP-L, a transcription factor related to RBP-Jkappa.

RBP-Jkappa is a sequence-specific DNA binding protein which plays a central role in signalling downstream of the Notch receptor by physically interacting with its intracellular region. Although at least four Notch genes exist in mammals, it is unknown whether each Notch requires a specific downstream signalling molecule. Here we report isolation and characterization of a mouse RBP-Jkappa-related gene named RBP-L that is expressed almost exclusively in lung, in contrast to the ubiquitous expression of RBP-Jkappa. For simplicity, we propose to call RBP-Jkappa RBP-J. The RBP-L protein bound to a DNA sequence almost identical to that of RBP-J. Surprisingly, RBP-L did not interact with any of the known four mouse Notch proteins. Although we found that RBP-L and EBNA-2 cooperated in transcriptional activation, they did not show significantly strong protein-protein interaction that can be detected by several in vivo and in vitro assays. This is again in contrast to physical association of RBP-J with EBNA-2. Several models to explain functional interaction between RBP-L and EBNA-2 are discussed.

Both Epstein-Barr viral nuclear antigen 2 (EBNA2) and activated Notch1 transactivate genes by interacting with the cellular protein RBP-J kappa.

The Epstein-Barr viral nuclear antigen 2 (EBNA2) plays a key role during establishment and maintenance of B cell immortalization after Epstein-Barr virus (EBV) infection. EBNA2 acts as a transactivator of cellular and viral genes. We studied two EBNA2 regulated viral promoters (TP1 promoter and LMP/TP2 promoter) in detail to learn more about the molecular mechanisms of EBNA2-mediated transactivation. In both promoters we could identify at least one binding site for the cellular repressor protein RBP-J kappa. EBNA2 is tethered to the EBNA2 responsive promoter elements by interaction with this cellular protein. Although necessary, the binding of RBP-J kappa is not sufficient for EBNA2-mediated transactivation. At least two further cellular proteins, which are different in the studied promoters are important for efficient transactivation. The identification of RBP-J kappa as central mediator of EBNA2 transactivation suggested an interference of EBNA2 with the highly conserved Notch receptor signal transduction pathway. We could show that an activated form of the Notch receptor can transactivate a reporter construct containing a hexamer of the two RBP-J kappa binding sites of the TP1 promoter supporting the idea that EBNA2 acts as a functional equivalent of an activated Notch receptor.

Epstein-Barr virus latent membrane protein (LMP1) is not sufficient to maintain proliferation of B cells but both it and activated CD40 can prolong their survival.

Epstein-Barr virus (EBV) infects human primary B lymphocytes and induces and maintains proliferation of these cells efficiently in vitro. Mutants of Epstein-Barr virus which express EBV nuclear antigen 2 (EBNA2) in a conditional fashion allow dissection of individual contributions of viral genes to B cell immortalization. EBNA2 is a transcriptional activator of cellular and viral genes, including the viral latent membrane protein 1 (LMP1), which is essential for B cell immortalization and has oncogenic effects in non-lymphoid cells. To analyze the role of this gene in B cell immortalization, LMP1 was constitutively expressed in B cells infected with EBV carrying a conditional EBNA2 allele. In the absence of functional EBNA2, LMP1 was incapable of sustaining B cell proliferation in two independent assays but induced a phenotype consistent with prolonged cell viability. Activation of CD40 displayed a comparable phenotype. These data indicate that both CD40 activation and LMP1 expression may use a common pathway for B cell activation. Proliferation of human B cells, however, requires one or more additional signals triggered by EBNA2.

Mutational analysis of the Epstein-Barr virus nuclear antigen 2 by far-Western blotting and DNA-binding studies.

We have previously shown by far-Western blotting that the Epstein-Barr virus nuclear antigen 2 (EBNA-2) both binds to a cellular protein of 130 kDa and histone H1, with the complex between EBNA-2 and p130 being tighter than between EBNA-2 and histone H1. Here we demonstrate that the N terminus of EBNA-2, which was previously shown to be necessary for transformation of B lymphocytes by EBNA-2, is essential for binding to p130. We further show data indicating that the binding of EBNA-2 to histone H1 appears not to be mediated exclusively via the basic Arg-Gly rich region in the C-terminal part of EBNA-2. With a MAb directed against the Trp-Trp322-Pro (WWP) motif of EBNA-2, which is known to be essential for the interaction of EBNA-2 with the cellular factor RBPJkappa/CBF1, we could inhibit the DNA binding of EBNA-2, providing further evidence that this region of EBNA-2 forms direct contact with RBPJkappa/CBF1.

Epstein-Barr virus nuclear antigen 2 (EBNA2)-oestrogen receptor fusion proteins complement the EBNA2-deficient Epstein-Barr virus strain P3HR1 in transformation of primary B cells but suppress growth of human B cell lymphoma lines.

To develop a transformation system with a conditional Epstein-Barr virus nuclear antigen 2 (EBNA2) gene, we fused the hormone binding domain of the oestrogen receptor to the N or C terminus of EBNA2. In promoter transactivation as well as primary B cell transformation assays these chimeric EBNA2 proteins are able to substitute for wild-type EBNA2 in the presence of oestrogen. Here we provide evidence that this transformation is the result of double infection of a cell with two virions, the P3HR1 virus genome and a mini-EBV plasmid carrying the chimeric EBNA2 gene. Unexpectedly, expression of the same EBNA2-oestrogen receptor fusion protein in established human B cell lymphoma lines resulted in growth retardation or growth arrest upon the addition of oestrogen. By titrating the oestrogen concentration in these stably transfected cells, the growth retarding and the transactivating function of the chimeric proteins could not be dissociated. We propose that growth inhibition of established B cell lymphoma lines is a novel function of EBNA2 which has not been detected in the absence of an inducible system. It remains open whether the growth retarding property of the EBNA2-oestrogen receptor fusion protein in B cell lymphoma lines is due to unphysiologically high expression of the chimeric protein or to interference with a cellular programme driving proliferation in these cell lines.

Epstein-Barr virus nuclear antigen 2 is a transcriptional suppressor of the immunoglobulin mu gene: implications for the expression of the translocated c-myc gene in Burkitt's lymphoma cells.

A conditional mutant of Epstein-Barr virus nuclear antigen 2 (EBNA2) regulated by estrogen was employed to study the effect of EBNA2 on the cellular phenotype. Activation of EBNA2 in lymphoblastoid cell lines (LCLs) and in B cell lymphoma lines resulted in down-regulation of cell surface IgM and Ig-mu steady-state RNA expression. In LCLs, activation of EBNA2 is required for maintaining proliferation, whereas in Burkitt's lymphoma (BL) cell lines with t(8;14) translocations, activation of EBNA2 induces growth arrest. In these cells, Northern and nuclear run-on analyses revealed rapid simultaneous repression of Ig-mu and c-myc transcription as early as 30 min after activation of EBNA2. Since c-myc expression is under the control of the Ig heavy chain locus in BL cell lines with a t(8;14) translocation, we propose that Ig-mu and c-myc are down-regulated by EBNA2 through a common mechanism.

Epstein-Barr virus nuclear antigen 2-estrogen receptor fusion proteins transactivate viral and cellular genes and interact with RBP-J kappa in a conditional fashion.

Epstein-Barr virus nuclear antigen 2 (EBNA2) is a transcriptional activator of viral and cellular genes involved in B cell transformation by EBV and is targeted to EBV responsive promoters through interaction with cellular DNA binding proteins such as RBP-J kappa. To develop a conditional system in which the function of EBNA2 can be switched on and off, we have fused the hormone binding domain of the estrogen receptor to the N- or C-terminus of EBNA2. Here we show that after transient or stable transfer of these chimerical EBNA2 genes into human B cell lymphoma lines, transactivation of LMP1, TP1, and TP2 promoter constructs, expression of the cell surface markers CD21 and CD23, and binding of EBNA2 to its cellular partner RBP-J kappa are dependent on the presence of estrogen. The EBNA2 fusion proteins proved to be virtually inactive in the absence of hormone.

Crucial sequences within the Epstein-Barr virus TP1 promoter for EBNA2-mediated transactivation and interaction of EBNA2 with its responsive element.

EBNA2 is one of the few genes of Epstein-Barr virus which are necessary for immortalization of human primary B lymphocytes. The EBNA2 protein acts as a transcriptional activator of several viral and cellular genes. For the TP1 promoter, we have shown previously that an EBNA2-responsive element (EBNA2RE) between -258 and -177 relative to the TP1 RNA start site is necessary and sufficient for EBNA2-mediated transactivation and that it binds EBNA2 through a cellular factor. To define the critical cis elements within this region, we cloned EBNA2RE mutants in front of the TP1 minimal promoter fused to the reporter gene for luciferase. Transactivation by EBNA2 was tested by transfection of these mutants in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR-1. The analysis revealed that two identical 11-bp motifs and the region 3' of the second 11-bp motif are essential for transactivation by EBNA2. Methylation interference experiments indicated that the same cellular factor in the absence of EBNA2 binds either one (complex I) or both (complex III) 11-bp motifs with different affinities, giving rise to two different specific protein-DNA complexes within the left-hand 54 bp of EBNA2RE. A third specific complex was shown previously to be present only in EBNA2-expressing cells and to contain EBNA2. Analysis of this EBNA2-containing complex revealed the same protection pattern as for complex III, indicating that EBNA2 interacts with DNA through binding of the cellular protein to the 11-bp motifs. Mobility shift assays with the different mutants demonstrated that one 11-bp motif is sufficient for binding the cellular factor, whereas for binding of EBNA2 as well as for efficient transactivation by EBNA2, both 11-bp motifs are required.

DNA-binding studies of the Epstein-Barr virus nuclear antigen 2 (EBNA-2): evidence for complex formation by latent membrane protein gene promoter-binding proteins in EBNA-2-positive cell lines.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) protein is essential for the immortalization of human primary B cells by EBV. EBNA-2 trans-activates cellular and viral genes like CD23, c-fgr, latent membrane protein 1 (LMP1) and terminal protein 1 (TP1). Trans-activation of the TP1 promoter and of the BamHI C promoter has already been investigated in detail and appears to be mediated via protein-protein interactions and not by direct binding of EBNA-2 type A (of EBV type 1) to the DNA. EBNA-2 is able to trans-activate the expression of the LMP gene in several cell lines. Various reports have delineated the cis-acting elements of the LMP promoter through which EBNA-2 mediates trans-activation. To determine whether EBNA-2 also trans-activates the LMP promoter by protein-protein interactions, we performed a series of gel retardation assays and competition experiments with LMP promoter fragments of different sizes. We determined that the protein-binding region on the LMP promoter was within a 42 bp fragment encompassing nucleotides -135 to -176 relative to the LMP transcriptional start site. None of the DNA fragments investigated indicated interaction of EBNA-2 with the DNA via protein-protein interactions. No significant differences between EBNA-2-positive and EBNA-2-negative nuclear extracts could be seen in the gel retardation assay under conditions that clearly showed binding of EBNA-2A to the TP1 promoter. However, analysis of sucrose gradient fractions in the gel retardation assay provided evidence that the LMP promoter-binding proteins form a complex of higher M(r) in EBNA-2-positive cell extracts. These complexes were destroyed by detergent. We deduce from these results that EBNA-2-positive cells might indeed contain specific complexes bound to the LMP promoter which are, however, too labile to be detected in a standard gel retardation assay.

Identification and characterization of an Epstein-Barr virus nuclear antigen 2-responsive cis element in the bidirectional promoter region of latent membrane protein and terminal protein 2 genes.

Epstein-Barr virus (EBV) transforms resting B cells in vitro very efficiently. The nuclear viral protein EBV nuclear antigen 2 (EBNA2) is absolutely required for this process and also acts as a transcriptional activator of cellular and viral genes. As shown previously, EBNA2 transactivates the promoters of the viral latent membrane proteins. It interacts indirectly with an EBNA2-responsive cis element of the terminal protein 1 (TP1) promoter. To identify the sequences mediating EBNA2 transactivation of the bidirectional promoter region driving expression of the latent membrane proteins LMP and TP2 in opposite directions, we assayed the effects of EBNA2 on the activities of promoter deletion and site-directed mutants of TP2 and LMP promoter luciferase reporter gene constructs by cotransfections into EBNA2-negative Burkitt's lymphoma cells. We were able to delineate an 80-bp EBNA2-responsive region (EBNA2RE) between -232 and -152 relative to the LMP RNA start site which could also mediate EBNA2-dependent activation on a heterologous promoter. Sequences of 20 and 32 bp located at the 5' and 3' ends, respectively, of the EBNA2RE were both essential for EBNA2 responsiveness. Full transactivation of the LMP and TP2 promoters seemed to require 20 bp of 5' adjacent sequences in addition to the 80-bp element. Electrophoretic mobility shift assays revealed specific protein-DNA complexes formed at the EBNA2RE. Oligonucleotides from -181 to -152 and -166 to -132 relative to the LMP RNA start site visualized one B-cell and one B-cell-plus-HL60-specific retarded protein-DNA complex, respectively. Additionally, an oligonucleotide from -253 to -210 revealed two specific protein-DNA complexes with nuclear extracts from different B and non-B cells, suggesting also the binding of ubiquitously expressed proteins on the EBNA2RE. Thus, these experiments defined a 80-bp cis element sufficient for conferring EBNA2 inducibility and demonstrated specific interactions of cellular proteins at DNA sequences within the EBNA2RE, which are critical for transactivation by EBNA2.