Gemma L. Kelly

Epstein-Barr virus-associated Burkitt lymphomagenesis selects for downregulation of the nuclear antigen EBNA2

Epstein–Barr virus (EBV) is etiologically linked to endemic Burkitt lymphoma (BL), but its contribution to lymphomagenesis, versus that of the chromosomal translocation leading to c-myc gene deregulation, remains unclear. The viruss growth-transforming (Latency III) program of gene expression is extinguished in tumor cells, and only a single viral protein, the EBV nuclear antigen (EBNA)1, is expressed via the alternative Latency I program. It is not known if BL arises from a B-cell subset in which EBV naturally adopts a Latency I infection or if a clone with limited antigen expression has been selected from an EBV-transformed Latency III progenitor pool. Here we identify a subset of BL tumors in which the Latency III-associated EBNA promoter Wp is active and most EBNAs are expressed, but where a gene deletion has specifically abrogated the expression of EBNA2. This implies that BL can be selected from a Latency III progenitor and that the principal selection pressure is for downregulation of the c-Myc antagonist EBNA2.

The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models.

Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.

An Epstein-Barr virus anti-apoptotic protein constitutively expressed in transformed cells and implicated in burkitt lymphomagenesis: the Wp/BHRF1 link.

Two factors contribute to Burkitt lymphoma (BL) pathogenesis, a chromosomal translocation leading to c-myc oncogene deregulation and infection with Epstein-Barr virus (EBV). Although the virus has B cell growth–transforming ability, this may not relate to its role in BL since many of the transforming proteins are not expressed in the tumor. Mounting evidence supports an alternative role, whereby EBV counteracts the high apoptotic sensitivity inherent to the c-myc–driven growth program. In that regard, a subset of BLs carry virus mutants in a novel form of latent infection that provides unusually strong resistance to apoptosis. Uniquely, these virus mutants use Wp (a viral promoter normally activated early in B cell transformation) and express a broader-than-usual range of latent antigens. Here, using an inducible system to express the candidate antigens, we show that this marked apoptosis resistance is mediated not by one of the extended range of EBNAs seen in Wp-restricted latency but by Wp-driven expression of the viral bcl2 homologue, BHRF1, a protein usually associated with the virus lytic cycle. Interestingly, this Wp/BHRF1 connection is not confined to Wp-restricted BLs but appears integral to normal B cell transformation by EBV. We find that the BHRF1 gene expression recently reported in newly infected B cells is temporally linked to Wp activation and the presence of W/BHRF1-spliced transcripts. Furthermore, just as Wp activity is never completely eclipsed in in vitro–transformed lines, low-level BHRF1 transcripts remain detectable in these cells long-term. Most importantly, recognition by BHRF1-specific T cells confirms that such lines continue to express the protein independently of any lytic cycle entry. This work therefore provides the first evidence that BHRF1, the EBV bcl2 homologue, is constitutively expressed as a latent protein in growth-transformed cells in vitro and, in the context of Wp-restricted BL, may contribute to virus-associated lymphomagenesis in vivo.

Three restricted forms of Epstein–Barr virus latency counteracting apoptosis in c-myc-expressing Burkitt lymphoma cells

Epstein–Barr virus (EBV), a human herpesvirus, transforms B cell growth in vitro through expressing six virus-coded Epstein–Barr nuclear antigens (EBNAs) and two latent membrane proteins (LMPs). In many EBV-associated tumors, however, viral antigen expression is more restricted, and the aetiological role of the virus is unclear. For example, endemic Burkitt lymphoma (BL) classically presents as a monoclonal, c-myc-translocation-positive tumor in which every cell carries EBV as an EBNA1-only (Latency I) infection; such homogeneity among EBV-positive cells, and the lack of EBV-negative comparators, hampers attempts to understand EBVs role in BL pathogenesis. Here, we describe an endemic BL that was unusually heterogeneous at the single-cell level and, in early passage culture, yielded a range of cellular clones, all with the same c-myc translocation but differing in EBV status. Rare EBV-negative cells were isolated alongside EBV-positive cells displaying one of three forms of restricted latency: (i) conventional Latency I expressing EBNA1 only from a WT virus genome, (ii) Wp-restricted latency expressing EBNAs 1, 3A, 3B, 3C, and -LP only from an EBNA2-deleted genome, and (iii) a previously undescribed EBNA2+/LMP1− latency in which all six EBNAs are expressed again in the absence of the LMPs. Interclonal comparisons showed that each form of EBV infection was associated with a specific degree of protection from apoptosis. Our work suggests that EBV acts as an antiapoptotic rather than a growth-promoting agent in BL by selecting among three transcriptional programs, all of which, unlike the full virus growth-transforming program, remain compatible with high c-myc expression.

Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Gene Deletion Is Consistently Linked with EBNA3A, -3B, and -3C Expression in Burkitt's Lymphoma Cells and with Increased Resistance to Apoptosis

ABSTRACT Most Epstein-Barr virus (EBV)-positive Burkitts lymphomas (BLs) carry a wild-type EBV genome and express EBV nuclear antigen 1 (EBNA1) selectively from the BamHI Q promoter (latency I). Recently we identified a distinct subset of BLs carrying both wild-type and EBNA2 gene-deleted (transformation-defective) viral genomes. The cells displayed an atypical “BamHI W promoter (Wp)-restricted” form of latency where Wp (rather than Qp) was active and EBNA1, -3A, -3B, -3C, and -LP were expressed in the absence of EBNA2 or latent membrane proteins 1 and 2. Here we present data strongly supporting the view that the EBNA2-deleted genome is transcriptionally active in these cells and the wild-type genome is silent. Single-cell cloning of three parental Wp-restricted BL lines generated clones carrying either both viral genomes or the EBNA2-deleted genome only, never clones with the wild-type genome only. All rescued clones displayed the Wp-restricted form of latency characteristic of the parent line and retained the original parent cell phenotype. Interestingly, Wp-restricted parent lines and derived clones were markedly more resistant to inducers of apoptosis than standard latency I BL lines. Furthermore, in vitro infection of EBV-negative BL lines with an EBNA2 gene-deleted virus generated EBV-positive converts with Wp-restricted latency and a similarly marked apoptosis resistance. We postulate that, in the subset of BLs displaying Wp-restricted latency, infection of a tumor progenitor cell with an EBNA2 gene-deleted virus has provided that cell with a survival advantage through broadening antigen expression to include the EBNA3 proteins.

Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53

The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

Quantitative Studies of Epstein-Barr Virus-Encoded MicroRNAs Provide Novel Insights into Their Regulation

ABSTRACT Epstein-Barr virus (EBV) has been shown to encode at least 40 microRNAs (miRNAs), an important class of molecules that negatively regulate the expression of many genes through posttranscriptional mechanisms. Here, we have used real-time PCR assays to quantify the levels of EBV-encoded BHRF1 and BART miRNAs in latently infected cells and in cells induced into the lytic cycle. During latency, BHRF1 miRNAs were seen only in cells with detectable Cp- and/or Wp-initiated EBNA transcripts, while the BART miRNAs were expressed in all forms of latent infection. Surprisingly, levels of different BART miRNAs were found to vary up to 50-fold within a cell line. However, this variation could not be explained by differential miRNA turnover, as all EBV miRNAs appeared to be remarkably stable. Following entry into the virus lytic cycle, miR-BHRF1-2 and -1-3 were rapidly induced, coincident with the onset of lytic BHRF1 transcripts, while miR-BHRF1-1 expression was delayed until 48 h and correlated with the appearance of Cp/Wp-initiated EBNA transcripts. In contrast, levels of BART miRNAs were relatively unchanged during virus replication, despite dramatic increases in BART transcription. Finally, we show that BHRF1 and BART miRNAs were delayed relative to the induction of BHRF1 and BART transcripts in freshly infected primary B cell cultures. In summary, our data show that changes in BHRF1 and BART transcription are not necessarily reflected in altered miRNA levels, suggesting that miRNA maturation is a key step in regulating steady-state levels of EBV miRNAs.

Burkitt's lymphoma: The Rosetta Stone deciphering Epstein-Barr virus biology

Epstein-Barr virus was originally identified in the tumour cells of a Burkitts lymphoma, and was the first virus to be associated with the pathogenesis of a human cancer. Studies on the relationship of EBV with Burkitts lymphoma have revealed important general principles that are relevant to other virus-associated cancers. In addition, the impact of such studies on the knowledge of EBV biology has been enormous. Here, we review some of the key historical observations arising from studies on Burkitts lymphoma that have informed our understanding of EBV, and we summarise the current hypotheses regarding the role of EBV in the pathogenesis of Burkitts lymphoma.

The essential role of evasion from cell death in cancer

The link between evasion of apoptosis and the development of cellular hyperplasia and ultimately cancer is implicitly clear if one considers how many cells are produced each day and, hence, how many cells must die to make room for the new ones (reviewed in Raff, 1996). Furthermore, cells are frequently experiencing noxious stimuli that can cause lesions in their DNA and faults in DNA replication can occur during cellular proliferation. Such DNA damage needs to be repaired efficiently or cells with irreparable damage must be killed to prevent subsequent division of aberrant cells that may fuel tumorigenesis (reviewed in Weinberg, 2007). The detection of genetic lesions in human cancers that activate prosurvival genes or disable proapoptotic genes have provided the first evidence that defects in programmed cell death can cause cancer (Tagawa et al., 2005; Tsujimoto et al., 1984; Vaux, Cory, and Adams, 1988) and this concept was proven by studies with genetically modified mice (Egle et al., 2004b; Strasser et al., 1990a). It is therefore now widely accepted that evasion of apoptosis is a requirement for both neoplastic transformation and sustained growth of cancer cells (reviewed in Cory and Adams, 2002; Hanahan and Weinberg, 2000; Weinberg, 2007). Importantly, apoptosis is also a major contributor to anticancer therapy-induced killing of tumor cells (reviewed in Cory and Adams, 2002; Cragg et al., 2009). Consequently, a detailed understanding of apoptotic cell death will help to better comprehend the complexities of tumorigenesis and should assist with the development of improved targeted therapies for cancer based on the direct activation of the apoptotic machinery (reviewed in Lessene, Czabotar, and Colman, 2008).

Genetic analysis of the Epstein-Barr virus-coded leader protein EBNA-LP as a co-activator of EBNA2 function

Co-operation between the Epstein-Barr virus (EBV)-coded leader protein EBNA-LP and the nuclear antigen EBNA2 appears to be critical for efficient virus-induced B cell transformation. Here we report the genetic analysis of EBNA-LP function using two transient co-transfection assays of co-operativity, activation of latent membrane protein 1 (LMP1) expression from a resident EBV genome in Akata-BL cells and activation of an EBNA2-responsive reporter construct. Small deletions were introduced into each of five conserved regions (CRs) of EBNA-LP sequence present in type 1 and type 2 EBV strains and in several primate lymphocryptovirus EBNA-LP homologues. Deletions within all three CRs in the EBNA-LP W1W2 repeat domain completely abrogated function, through inhibition of nuclear localization in the cases of CR1 and CR2 but not of CR3; deletions within CR4 and CR5 in the Y1Y2 unique domain had relatively little effect, yet loss of the whole Y2 sequence blocked activity. Alanine substitution of serine residues within potential phosphorylation sites identified two mutants of particular interest. Substitution of three such residues (S34,36,63) within W1W2 not only abrogated EBNA-LP activity but was associated with a complete loss of EBNA2 detectability in co-transfected cells, implying possible destabilization of the co-expressed EBNA2 protein. More importantly the individual substitution of S36 completely blocked EBNA-LP/EBNA2 co-operativity while retaining EBNA2 expression. We infer critical roles for the CR3 domain and for the S36 residue in EBNA-LPs co-operative function.