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

e Glasgow and Manchester Experimental Cancer Medicine Centres (ECMC), which are funded by CR-UK and the Chief Scientist’s Office (Scotland). We acknowledge the funders who have contributed to this work: MRC stratified medicine infrastructure award (A.D.W.), CR-UK C11074/A11008 (F.P., L.E.M.H., T.L.H., A.D.W.); LLR08071 (S.A.A., E.C.); LLR11017 (M.C.); SCD/04 (M.C.); LLR13035 (S.A.A., K.D., A.D.W., and A.P.); LLR14005 (M.T.S., D.V.); KKL690 (L.E.P.); KKL698 (P.B.); LLR08004 (A.D.W., A.P. and A.J.W.); MRC CiC (M.E.D.); The Howat Foundation (FACS support); Friends of Paul O’Gorman (K.D. and FACS support); ELF 67954 (S.A.A.); BSH start up fund (S.A.A.); MR/K014854/1 (K.D.)

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

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

CXCR2 and CXCL4 regulate survival and self-renewal of hematopoietic stem/progenitor cells

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 hematopoietic stem/progenitor cells, revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, CXCL13) were up-regulated in human quiescent CD34+Hoescht-Pyronin Y- and primitive CD34+38-, as compared to proliferating CD34+Hoechst+Pyronin Y+ and CD34+38+ stem/progenitor cells. This suggested that chemokines may play an important role in the homeostasis of HSCs. In human CD34+ hematopoietic cells, knock-down of CXCL4 or pharmacological 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 HSC, hematopoietic progenitor cell (HPC)-1, HPC-2 and Lin-Sca-1+c-Kit+ sub-populations. 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 knock-down 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 hematopoietic stem/progenitor cell fates, including survival and self-renewal

EBNA1 Induces B-cells to Respond to IL-2 Survival Signals

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

EBNA1 Induces B-cells to Respond to IL-2 Survival Signals

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

187-OR: Declines in ß-Cell Glucose Responsiveness Correlate with Loss of Glucose Metabolism during T1D Pathogenesis

Features of type 1 diabetes (T1D) include loss of first-phase insulin secretion in response to glucose, declining c-peptide, and glucose intolerance; indicating β cell dysfunction. Here, we investigate the mechanisms of this dysfunction with the hypothesis that immune dysregulation during the early stages of T1D development impacts β cell dysfunction in pathways affecting glucose metabolism. The live pancreas tissue slice (LPTS) model is ideal for the study of T1D pathogenesis due to its preservation of the pancreatic environment. LPTS were generated from organ donors without diabetes or autoantibodies (ND, n=9) , donors positive for one or more autoantibodies without a diagnosis of T1D (AAb+, n=6) , and donors with T1D (T1D, n=4) . Imaging studies were conducted to assess the impact of T cell infiltration on β cell function through Ca2+ imaging and slice perifusion. Islets from ND and AAb+ donors had no insulitis and exhibited Ca2+ responses to both high glucose (HG) and potassium chloride (KCl) . A majority of β cell+ islets in tissue from donors with T1D had infiltrating T cells with several having insulitis. Islet HG response from donors with T1D was heterogenous (∼50% failed to respond) while all T1D islets exhibited responses to KCl. Insulin secretion was significantly higher in LPTS from ND donors than in donors with T1D (p=0.0079) . To determine the basis for this loss of function, we assessed transcriptomics of islets within these cohorts. When comparing gene expression levels in islets from ND versus T1D, significant differences were observed in genes involved in glycolysis (GAPDH, p=3.27x10-21) , the citric acid cycle components (OGDH, p=1.88x10-18) , and the electric transport chain (F1FoATP synthase, p=2.69x10-13) . The decreased expression of glucose metabolism genes during T1D development along with the loss of β cell function in LPTS demonstrates β cell dysfunction before their demise and may be a contributory mechanism towards the pathogenesis of disease. Disclosure M.Huber: None. M.Slak rupnik: None. D.M.Drotar: None. H.Hiller: None. M.Beery: None. I.Kusmartseva: None. M.A.Atkinson: None. E.Phelps: Research Support; Immunocore, Ltd. C.E.Mathews: None. Funding National Institutes of Health (5T32DK108736-03) National Institutes of Health (PO1 AI42288) </jats:sec