RESUMEN
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.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Leucemia Mieloide , Humanos , División Celular , Células Madre Embrionarias , Neoplasia Residual , Proteína p53 Supresora de Tumor/genéticaRESUMEN
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.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Linfoma/virología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Animales , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/metabolismo , Muerte Celular/fisiología , Línea Celular , Factor de Transcripción E2F1/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Humano 4/metabolismo , Humanos , Interleucina-2/metabolismo , Linfoma/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
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.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Acetamidas/farmacología , Acetamidas/uso terapéutico , Animales , Antígenos CD34/metabolismo , Azepinas/farmacología , Azepinas/uso terapéutico , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Femenino , Proteínas de Fusión bcr-abl/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Imidazolinas/farmacología , Imidazolinas/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Masculino , Ratones , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/trasplante , Proteómica , Proteínas Proto-Oncogénicas c-myc/deficiencia , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Transcriptoma , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
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.
Asunto(s)
Proliferación Celular/fisiología , Células Madre Hematopoyéticas/metabolismo , Factor Plaquetario 4/metabolismo , Receptores de Interleucina-8B/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Supervivencia Celular/fisiología , Femenino , Células Madre Hematopoyéticas/citología , Humanos , Masculino , Ratones , Ratones Noqueados , Receptores de Interleucina-8B/genética , Bazo/citología , Bazo/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Senescencia Celular/fisiología , Chaperonas de Histonas/metabolismo , Factores de Transcripción/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular , Senescencia Celular/genética , Cromatina/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos , Chaperonas de Histonas/genética , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Ratones , Papiloma/patología , Neoplasias Cutáneas/patología , Tamoxifeno/farmacología , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Senescencia Celular/genética , Epigénesis Genética , Neoplasias/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Islas de CpG , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Expresión Génica , Genoma Humano , Humanos , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Transporte de ProteínasRESUMEN
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.
Asunto(s)
Senescencia Celular/fisiología , Melanocitos/fisiología , Melanoma/etiología , Nevo/fisiopatología , Vía de Señalización Wnt/fisiología , Animales , Línea Celular Tumoral , Cartilla de ADN/genética , Células HEK293 , Humanos , Immunoblotting , Inmunohistoquímica , Melanocitos/citología , Ratones , Análisis por Micromatrices , Nevo/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARNRESUMEN
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.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Herpesvirus Humano 4/inmunología , Interleucina-15/farmacología , Interleucina-2/farmacología , Linfocitos/citología , Linfocitos/inmunología , Animales , Antígenos CD/inmunología , Linfocitos B/inmunología , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Genoma Viral , Humanos , Interleucina-15/biosíntesis , Interleucina-2/biosíntesis , Linfocitos/efectos de los fármacos , Linfoma/patología , Linfoma/virología , Ratones , Ratones Transgénicos , Neoplasias/virología , Linfocitos T/inmunología , Timidina/metabolismoRESUMEN
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).
Asunto(s)
Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Herpesvirus Humano 4/fisiología , Linfoma de Células B/virología , Proteínas Proto-Oncogénicas c-myc/fisiología , Animales , Linfocitos B/virología , Southern Blotting , Western Blotting , Cromosomas/genética , Cruzamientos Genéticos , Infecciones por Virus de Epstein-Barr/patología , Femenino , Amplificación de Genes , Reordenamiento Génico , Humanos , Cariotipificación , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Linfoma de Células T/química , Linfoma de Células T/patología , Linfoma de Células T/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Translocación Genética , TrisomíaRESUMEN
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.