Activating p53 abolishes self-renewal of quiescent leukaemic stem cells in residual CML disease.

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.

Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells.

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.

HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia.

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.

Senescent cells harbour features of the cancer epigenome.

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.

Wnt signaling potentiates nevogenesis.

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 senescence-associated 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 and malignant potential. In a mouse, activated Wnt signaling delays senescence-associated 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.

Epstein-Barr virus nuclear antigen-1 renders lymphocytes responsive to IL-2 but not IL-15 for survival.

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.

Epstein-Barr virus nuclear antigen-1 and Myc cooperate in lymphomagenesis.

The lymphomagenic action of myc genes in conjunction with Epstein-Barr virus nuclear antigen-1 (EBNA-1) have been examined using transgenic mice in several separate tests. Synergy between Myc and EBNA-1 in lymphomagenesis was revealed in a cross breed study where co-expression of transgenic myc and EBNA-1 led to a tumor latency period reduced significantly in some crosses. In the resulting bitransgenic tumors, expression of the Emu-myc genes was not affected by EBNA-1 expression. MoMLV was utilized as a transposon tag to activate cellular oncogenes by infection of EmuEBNA-1 mice. Rearrangement at the c-myc locus in B cell tumors from these mice again suggests a cooperative action between myc and EBNA-1. Tumors arising in EmuEBNA-1 mice typically showed a trisomy of chromosome 15, upon which the c-myc locus resides. Bitransgenic tumors (EBNA-1/c-myc) did not show trisomy 15. This raises the possibility that amplification of c-myc is factorial in the selection of trisomy 15 in these tumors. These data indicate that myc and EBNA-1 act cooperatively and are not redundant in lymphomagenesis. Expression of EBNA-1 by EBV may provide a selection pressure in addition to translocation of the c-myc locus in the genesis of endemic Burkitt's lymphoma (BL).

bcl-xL and RAG genes are induced and the response to IL-2 enhanced in EmuEBNA-1 transgenic mouse lymphocytes.

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.