Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease - with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission.

Although it has been recognized for many years that cancer stem cells and embryonic stem cells (ESC) share molecular features, identifying ways to exploit this therapeutically has proved challenging. To date, these shared features have not been examined in the leukemic stem cells (LSC) found in patients with chronic myeloid leukemia (CML).

Epstein–Barr virus (EBV)-associated Burkitt’s lymphoma is characterised by the deregulation of c-Myc expression and a restricted viral gene expression pattern in which the EBV nuclear antigen-1 (EBNA1) is the only viral protein to be consistently expressed. EBNA1 is required for viral genome propagation and segregation during latency. However, it has been much debated whether the protein plays a role in viral-associated tumourigenesis. We show that the lymphomas which arise in EµEBNA1 transgenic mice are unequivocally linked to EBNA1 expression and that both C-Myc and Mdm2 deregulation are central to this process. Tumour cell survival is supported by IL-2 and there is a skew towards CD8-positive T cells in the tumour environment, while the immune check-point protein PD-L1 is upregulated in the tumours. Additionally, several isoforms of Mdm2 are upregulated in the EµEBNA1 tumours, with increased phosphorylation at ser166, an expression pattern not seen in Eµc-Myc transgenic tumours. Concomitantly, E2F1, Xiap, Mta1, C-Fos and Stat1 are upregulated in the tumours. Using four independent inhibitors of Mdm2 we demonstrate that the EµEBNA1 tumour cells are dependant upon Mdm2 for survival (as they are upon c-Myc) and that Mdm2 inhibition is not accompanied by upregulation of p53, instead cell death is linked to loss of E2F1 expression, providing new insight into the underlying tumourigenic mechanism. This opens a new path to combat EBV-associated disease.

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Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show-using proteomics, transcriptomics and network analyses-that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated.

The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34(+)Hoescht(-)Pyronin Y- and primitive CD34(+)38(-), as compared with proliferating CD34(+)Hoechst(+)Pyronin Y+ and CD34(+)38(+) stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34(+) hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2(-/-) mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin(-)Sca-1(+)c-Kit(+) subpopulations. Cxcr2(-/-) stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit(+) cells, and Cxcl4(-/-) mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal.

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression.

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway (senescence-associated secretory phenotype). Cellular senescence is also a tumor suppressor mechanism, to which both proliferation arrest and senescence-associated secretory phenotype are thought to contribute. The melanocytes within benign human nevi are a paradigm for tumor-suppressive senescent cells in a premalignant neoplasm. Here a comparison of proliferating and senescent melanocytes and melanoma cell lines by RNA sequencing emphasizes the importance of senescenceassociated proliferation arrest in suppression of transformation. Previous studies showed that activation of the Wnt signaling pathway can delay or bypass senescence. Consistent with this, we present evidence that repression of Wnt signaling contributes to melanocyte senescence in vitro. Surprisingly, Wnt signaling is active in many senescent human melanocytes in nevi, and this is linked to histological indicators of higher proliferative andmalignant potential. In a mouse, activated Wnt signaling delays senescenceassociated proliferation arrest to expand the population of senescent oncogene-expressing melanocytes. These results suggest that Wnt signaling can potentiate nevogenesis in vivo by delaying senescence. Further, we suggest that activated Wnt signaling in human nevi undermines senescence-mediated tumor suppression and enhances the probability of malignancy.

Altered DNA methylation and associated destabilization of genome integrity and function is a hallmark of cancer. Replicative senescence is a tumour suppressor process that imposes a limit on the proliferative potential of normal cells that all cancer cells must bypass. Here we show by whole-genome single-nucleotide bisulfite sequencing that replicative senescent human cells exhibit widespread DNA hypomethylation and focal hypermethylation. Hypomethylation occurs preferentially at gene-poor, late-replicating, lamin-associated domains and is linked to mislocalization of the maintenance DNA methyltransferase (DNMT1) in cells approaching senescence. Low-level gains of methylation are enriched in CpG islands, including at genes whose methylation and silencing is thought to promote cancer. Gains and losses of methylation in replicative senescence are thus qualitatively similar to those in cancer, and this 'reprogrammed' methylation landscape is largely retained when cells bypass senescence. Consequently, the DNA methylome of senescent cells might promote malignancy, if these cells escape the proliferative barrier.

Epstein-Barr virus nuclear antigen-1 (EBNA-1) is the only latent protein expressed in all virus-associated tumours. It plays a critical role in viral propagation and in the replication, episomal maintenance and partitioning of the viral genome. However, its tumorigenic potential is debated. We have previously shown that lymphocytes from a tumour-prone, EBNA-1-expressing, transgenic mouse line show increased responsiveness to interleukin-2 (IL-2). It was important to determine whether this property was unique to the transgenic line or whether it is a general consequence of EBNA-1 expression in B cells. In order to distinguish between these possibilities, explanted lymphocytes from two independent transgenic mouse lines were examined. The lymphocytes from both lines showed enhanced proliferation rates compared with controls. The transgenic lymphocytes survived for extended periods in culture, dependent on the dose of IL-2, while IL-15 (the receptor of which shares the beta and gamma chain components of the IL-2 receptor) induced little effect. In accordance with this, transgenic B cells showed enhanced induction of expression of the IL-2 receptor alpha chain (CD25), which modulates affinity for the ligand. As this phenotype is evident in lymphocytes from mice of both lines, it is necessarily independent of any transgene insertion site effects and may be attributed to EBNA-1 expression. Furthermore, 10/12 tumour-bearing transgenic mice had elevated IL-2 levels in serum and 4/6 tumours were CD25 positive. IL-2 is normally produced by activated T cells in vivo; thus, chronic immune activation or modulation could elicit this unique mode of virus-infected cell survival.

Background: There is growing evidence that EBNA-1 can influence B-cell survival, but whether this is sufficient to predispose transgenic mice to lymphomagenesis is controversial. We have previously shown that two out of 12 established transgenic mouse lines generated using an EµEBNA-1 transgene expressed EBNA-1 (Wilson et al., 1996, EMBO J., 15, p3117). First, this demonstrated that in vivo expression of full length EBNA-1 from a relatively simple transgene is not efficient. Second, mice of the two expressing lines succumb to B-cell lymphoma with identical pathology, but with dramatically different penetrance and latency to onset, essentially a fast tumour line (line 26) and a slow tumour line (line 59). A further development from the “fast line” was that a spontaneous partial transgene deletion arose, giving rise to a sub-line of mice (designated 26A), which no longer showed EBNA-1 expression or developed lymphoma. Methods: In order to explore the contribution of EBNA-1 to the phenotype and to examine if there is any influence from cellular sequences at the sites of transgene insertion, we have taken three approaches: [1] To explore the phenotype of lymphocytes from both transgenic lines, prior to tumour development, where any characteristic in common between the two lines must result from EBNA-1 expression; [2] To use dominant negative EBNA-1 expression to examine the effect of EBNA-1 “withdrawal”; [3] To identify and characterise the transgene insertion sites. Results: [1] Lymphocytes explanted and cultured from mice of both transgenic lines initially show enhanced proliferation and then prolonged survival compared to non-transgenic wild-type sibling controls. This property is only evident when the cells are cultured in the presence of interleukin-2 (IL-2). [2] Transfection and expression of dominant negative forms of EBNA-1 in a cell line derived from an EBNA-1 expressing line 59 tumour (co-expressing LMP1) is not compatible with the survival of these cells, while expression in cell lines derived from LMP1-only tumours is innocuous. [3] The transgene insertion site for line 59 has been precisely mapped to murine chromosome 4 band D3. No known oncogenes lie withing a Mb region encompassing the transgene. The transgene insertion site for sub-line 26A has been precisely mapped to murine chromosome 3 band H2. No known oncogenes lie within a Mb region encompassing the transgene. The transgene insertion site for line 26 has been mapped to murine chromosome 5 band B and fine mapping is ongoing. Conclusion: EBNA-1 promotes lymphocyte survival in the transgenic system.

The latent membrane proteins (LMP1 and LMP2) of Epstein-Barr virus (EBV) both impinge upon multiple cellular signalling pathways. LMP1, a viral onco-protein, can mimic a constitutively activated CD40 receptor in culture, activating NF-kB and JNK. Using EµLMP1 transgenic mice we find that some CD40 activities can be restored by LMP1 in CD40 null mice, in particular immunoglobulin isotype switching. We have previously shown that LMP2A allows the survival of B-cell receptor (BCR) negative B-cells in vivo (Caldwell et. al., 1998 Immunity 9:405-411) and have proposed this may be mediated by mimicking BCR survival signals. In support of this hypothesis, we will present data indicating that LMP2A expressing transgenic memory B-cells survive in the absence of antigen, using adoptive transfer experiments to CD40 null recipients. Furthermore, affinity maturation in the EµLMP2A mice may be impaired, again suggestive of BCR mediated survival instead of deletion of low affinity B-cells. Thus the activities of these two viral genes can promote B-cell survival and maturation, required in the EBV life cycle with the inadvertent consequence of predisposing the B-cell to lymphomagenesis.

Whether EBNA-1 contributes to tumourigenesis by means other than its function in viral DNA propagation is controversial. In order to address this, we have explored the consequences of EBNA-1 expression in the B-cells of two independently derived lines of transgenic mice (Wilson et al., 1996, EMBO J., 15, p3117). With the aim of examining the direct effects of EBNA-1 expression and not secondary mutations occurring through tumourigenesis, our studies have been conducted using explanted transgenic lymphocytes prior to the development of any tumour pathology. Transgenic lymphocytes show enhanced proliferation compared to controls and prolonged survival when cultured in the presence of IL-2. Surviving cells are B-cells and continue to express EBNA-1. This phenotype is demonstrated by lymphocytes derived from both transgenic mouse lines, developed independently and with distinct transgene integration events. As such the properties of prolonged cell survival and enhanced growth cannot be due to insertion site effects and can only be attributed to the actions of EBNA-1. These properties are characteristic oncogenic activities and in this system are context dependant, upon IL-2 signalling, a cytokine normally produced by activated T-cells supporting both T- and B-cell immune responses. In order to explore the consequences upon tumour development of co-expression of EBNA-1 (inducing B-cell responsiveness to IL-2 as described above), LMP1 (a partial mimic of CD40 constitutive signalling) and LMP2A (mimicking B-cell receptor survival signals) a tritransgenic mouse crossbreed was developed. As previously reported, EµEBNA-1 mice develop B-cell tumours. Co-expression of EBNA-1 and LMP1 showed no impact upon the latency to tumour development while co-expression of EBNA-1 and LMP2A showed a slight inhibition in tumour development. However, co-expression of all three latent proteins significantly delayed the EBNA-1 induced tumour onset. Whether together the LMPs activate opposing mechanisms to EBNA-1 in B-cell development and differentiation will be discussed.

We have described transgenic mice expressing Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) in B-cells which show a predisposition to lymphoma. To investigate the underlying oncogenic mechanisms, we have cross bred transgenic strains of mice, examined the pre-tumour B-cell phenotype and investigated the expression levels of selected cellular genes as a response to EBNA-1 expression. We have found that bcl-xL and the recombination activating genes (RAG) 1 and 2 are induced in pre-neoplastic samples of EBNA-1 expressing mice. Induction of bcl-xL may explain the observed redundancy in lymphomagenesis between transgenic EBNA-1 and bcl-2. In addition, bone marrow cells derived from the EmuEBNA-1 mice show a greater capacity for cultured growth compared to controls, particularly in the presence of IL-2. Notably, bcl-xL expression is responsive to IL-2. These data shed new light on the potential contribution of EBNA-1 to EBV associated tumorigenicity as well as to the viral life cycle and open a potential avenue for therapeutic intervention.