Asunto(s)
Linfoma de Burkitt , Herpesvirus Humano 4 , Factores de Transcripción SOXC , Adulto , Femenino , Humanos , Masculino , Linfoma de Burkitt/patología , Linfoma de Burkitt/genética , Linfoma de Burkitt/virología , Linfoma de Burkitt/diagnóstico , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/patología , Herpesvirus Humano 4/aislamiento & purificación , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismoRESUMEN
Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50-100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers. IMPORTANCE: The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr , Proteínas F-Box , Herpesvirus Humano 4 , Proteínas Proto-Oncogénicas c-bcl-6 , Humanos , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/genética , Línea Celular Tumoral , Linfocitos B/metabolismo , Linfocitos B/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Proteolisis , Proliferación Celular , Ubiquitinación , Linfoma de Burkitt/virología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , Antígenos Virales/metabolismo , Antígenos Virales/genética , Centro Germinal/metabolismo , Centro Germinal/virología , Proteína-Arginina N-MetiltransferasasRESUMEN
Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection. To gain insight into the host miRNAs regulating transitions from EBV latency into the lytic stage, we conducted a CRISPR/Cas9-based screen in EBV+ Burkitt lymphoma (BL) cells using anti-Ig antibodies to crosslink the B cell receptor (BCR) and induce reactivation. Using a gRNA library against >1500 annotated human miRNAs, we identified miR-142 as a key regulator of EBV reactivation. Genetic ablation of miR-142 enhanced levels of immediate early and early lytic gene products in infected BL cells. Ago2-PAR-CLIP experiments with reactivated cells revealed miR-142 targets related to Erk/MAPK signaling, including components directly downstream of the B cell receptor (BCR). Consistent with these findings, disruption of miR-142 enhanced SOS1 levels and Mek phosphorylation in response to surface Ig cross-linking. Effects could be rescued by inhibitors of Mek (cobimetinib) or Raf (dabrafenib). Taken together, these results show that miR-142 functionally regulates SOS1/Ras/Raf/Mek/Erk signaling initiated through the BCR and consequently, restricts EBV entry into the lytic cycle.
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Sistemas CRISPR-Cas , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , MicroARNs , Activación Viral , Latencia del Virus , Humanos , Herpesvirus Humano 4/genética , MicroARNs/genética , MicroARNs/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Linfoma de Burkitt/virología , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Línea Celular TumoralRESUMEN
Epstein-Barr virus (EBV) is a ubiquitous human tumor virus that establishes lifelong, persistent infections in B cells. The presence of EBV in cancer cells presents an opportunity to target these cells by reactivating the virus from latency. In this study, we developed a novel approach for EBV reactivation termed clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated EBV reactivation (CMER) strategy. Using modified CRISPR-associated protein 9 (dCas9) fused with VP64, we designed 10 single guide RNAs (sgRNAs) to target and activate the EBV immediate-early gene promoter. In Akata Burkitt lymphoma cells, 9 out of 10 CMER sgRNAs effectively reactivated EBV. Among these, CMER sgRNA-5 triggered robust reactivation across various cell types, including lymphoma, gastric cancer, and nasopharyngeal carcinoma cells. Importantly, the combination of CMER and ganciclovir selectively eliminated EBV-positive cells, regardless of their cell origin. These findings indicate that targeted virus reactivation by CMER, combined with nucleoside analog therapy, holds promise for EBV-associated cancer treatment. IMPORTANCE: This study explores a novel strategy called clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated Epstein-Barr virus (EBV) reactivation (CMER) to reactivate the Epstein-Barr virus in cancer cells. EBV is associated with various cancers, and reactivating EBV from latency offers a potential therapeutic strategy. We utilized an enzymatically inactive CRISPR-associated protein 9 (dCas9) fused with VP64 and designed 10 single guide RNAs to target the EBV immediate-early gene promoter. Nine of these sgRNAs effectively reactivated EBV in Burkitt lymphoma cells, with CMER sgRNA-5 demonstrating strong reactivation across different cancer cell types. Combining CMER with ganciclovir selectively eliminated EBV-positive cells, showing promise for EBV-associated cancer treatment.
Asunto(s)
Sistemas CRISPR-Cas , Infecciones por Virus de Epstein-Barr , Ganciclovir , Herpesvirus Humano 4 , Activación Viral , Humanos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/fisiología , Activación Viral/efectos de los fármacos , Activación Viral/genética , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/genética , Línea Celular Tumoral , Ganciclovir/farmacología , Latencia del Virus/genética , Latencia del Virus/efectos de los fármacos , Antivirales/farmacología , Linfoma de Burkitt/genética , Linfoma de Burkitt/virología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteína 9 Asociada a CRISPR/genéticaRESUMEN
Although the toxicity of arsenic depends on its chemical forms, few studies have taken into account the ambiguous phenomenon that sodium arsenite (NaAsO2) acts as a potent carcinogen while arsenic trioxide (ATO, As2O3) serves as an effective therapeutic agent in lymphoma, suggesting that NaAsO2 and As2O3 may act via paradoxical ways to either promote or inhibit cancer pathogenesis. Here, we compared the cellular response of the two arsenical compounds, NaAsO2 and As2O3, on the Burkitt lymphoma cell model, the Epstein Barr Virus (EBV)-positive P3HR1 cells. Using flow cytometry and biochemistry analyses, we showed that a NaAsO2 treatment induces P3HR1 cell death, combined with drastic drops in ΔΨm, NAD(P)H and ATP levels. In contrast, As2O3-treated cells resist to cell death, with a moderate reduction of ΔΨm, NAD(P)H and ATP. While both compounds block cells in G2/M and affect their protein carbonylation and lipid peroxidation, As2O3 induces a milder increase in superoxide anions and H2O2 than NaAsO2, associated to a milder inhibition of antioxidant defenses. By electron microscopy, RT-qPCR and image cytometry analyses, we showed that As2O3-treated cells display an overall autophagic response, combined with mitophagy and an unfolded protein response, characteristics that were not observed following a NaAsO2 treatment. As previous works showed that As2O3 reactivates EBV in P3HR1 cells, we treated the EBV- Ramos-1 cells and showed that autophagy was not induced in these EBV- cells upon As2O3 treatment suggesting that the boost of autophagy observed in As2O3-treated P3HR1 cells could be due to the presence of EBV in these cells. Overall, our results suggest that As2O3 is an autophagic inducer which action is enhanced when EBV is present in the cells, in contrast to NaAsO2, which induces cell death. That's why As2O3 is combined with other chemicals, as all-trans retinoic acid, to better target cancer cells in therapeutic treatments.
Asunto(s)
Trióxido de Arsénico , Arsenicales , Arsenitos , Autofagia , Mitocondrias , Estrés Oxidativo , Óxidos , Compuestos de Sodio , Trióxido de Arsénico/farmacología , Arsenitos/farmacología , Arsenitos/toxicidad , Humanos , Estrés Oxidativo/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Compuestos de Sodio/farmacología , Arsenicales/farmacología , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Óxidos/farmacología , Muerte Celular/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Herpesvirus Humano 4/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Peróxido de Hidrógeno/farmacología , Peroxidación de Lípido/efectos de los fármacos , Linfoma de Burkitt/virología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Linfoma de Burkitt/tratamiento farmacológicoRESUMEN
ABSTRACT: SRY-related HMG-box gene 11 (SOX11) is a transcription factor overexpressed in mantle cell lymphoma (MCL), a subset of Burkitt lymphomas (BL) and precursor lymphoid cell neoplasms but is absent in normal B cells and other B-cell lymphomas. SOX11 has an oncogenic role in MCL but its contribution to BL pathogenesis remains uncertain. Here, we observed that the presence of Epstein-Barr virus (EBV) and SOX11 expression were mutually exclusive in BL. SOX11 expression in EBV-negative (EVB-) BL was associated with an IGâ·MYC translocation generated by aberrant class switch recombination, whereas in EBV-negative (EBV-)/SOX11-negative (SOX11-) tumors the IGâ·MYC translocation was mediated by mistaken somatic hypermutations. Interestingly, EBV- SOX11-expressing BL showed higher frequency of SMARCA4 and ID3 mutations than EBV-/SOX11- cases. By RNA sequencing, we identified a SOX11-associated gene expression profile, with functional annotations showing partial overlap with the SOX11 transcriptional program of MCL. Contrary to MCL, no differences on cell migration or B-cell receptor signaling were found between SOX11- and SOX11-positive (SOX11+) BL cells. However, SOX11+ BL showed higher adhesion to vascular cell adhesion molecule 1 (VCAM-1) than SOX11- BL cell lines. Here, we demonstrate that EBV- BL comprises 2 subsets of cases based on SOX11 expression. The mutual exclusion of SOX11 and EBV, and the association of SOX11 with a specific genetic landscape suggest a role of SOX11 in the early pathogenesis of BL.
Asunto(s)
Linfoma de Burkitt , Herpesvirus Humano 4 , Factores de Transcripción SOXC , Humanos , Linfoma de Burkitt/genética , Linfoma de Burkitt/virología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismo , Herpesvirus Humano 4/genética , Regulación Neoplásica de la Expresión Génica , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/virología , Mutación , ADN Helicasas/genética , ADN Helicasas/metabolismo , Translocación Genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Masculino , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Proteínas NuclearesRESUMEN
Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.
Asunto(s)
Linfocitos B , Linfoma de Burkitt , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Proteínas Proto-Oncogénicas c-myc , Latencia del Virus , Animales , Linfoma de Burkitt/virología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Linfoma de Burkitt/genética , Humanos , Ratones , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Linfocitos B/virología , Linfocitos B/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Apoptosis , Proteínas Virales/metabolismo , Proteínas Virales/genéticaRESUMEN
The Epstein-Barr Virus (EBV) encodes viral microRNAs (miRs) that have been implicated in the pathogenesis of nasopharyngeal and gastric carcinomas, yet their potential roles in lymphomas remain to be fully elucidated. This study evaluated the impact of CRISPR/Cas9-mediated knockdown of EBV miRs BART-7 and BART-9 in EBV-positive Burkitt lymphoma cells Akata. As anticipated, the Akata cells subjected to CRISPR/Cas9-mediated knockdown of either EBV BART-7 or BART-9 exhibited a significant reduction in the expression of these viral miRs compared to cells with wild-type (wt) EBV genomes. This outcome effectively validates the experimental model employed in this study. Knocking down either BART-7 or BART-9 resulted in a notable reduction in cell viability and proliferation rates, alongside an elevation in the expression of EBV lytic genes. Global proteomic analysis revealed that the knockdown of EBV BART-7 significantly decreased the expression of ubiquitin/proteasome proteins while concurrently increasing RNA binding proteins (RBPs). Conversely, BART-9 knockdown reduced proteins associated with oxidoreductase activity, particularly those involved in fatty acid metabolism. Our findings unveil previously undiscovered EBV miRs BARTs 7 and 9 roles in cellular pathways relevant to both viral biology and lymphomagenesis.
Asunto(s)
Linfoma de Burkitt , Proliferación Celular , Herpesvirus Humano 4 , MicroARNs , ARN Viral , Humanos , Herpesvirus Humano 4/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/virología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , MicroARNs/genética , MicroARNs/metabolismo , Proliferación Celular/genética , Línea Celular Tumoral , ARN Viral/genética , Proteómica/métodos , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/metabolismoRESUMEN
Coinfection of human papillomavirus (HPV) and Epstein-Barr virus (EBV) has been detected in oropharyngeal squamous cell carcinoma. Although HPV and EBV replicate in differentiated epithelial cells, we previously reported that HPV epithelial immortalization reduces EBV replication within organotypic raft culture and that the HPV16 oncoprotein E7 was sufficient to inhibit EBV replication. A well-established function of HPV E7 is the degradation of the retinoblastoma (Rb) family of pocket proteins (pRb, p107, and p130). Here, we show that pRb knockdown in differentiated epithelia and EBV-positive Burkitt lymphoma (BL) reduces EBV lytic replication following de novo infection and reactivation, respectively. In differentiated epithelia, EBV immediate early (IE) transactivators were expressed, but loss of pRb blocked expression of the early gene product, EA-D. Although no alterations were observed in markers of epithelial differentiation, DNA damage, and p16, increased markers of S-phase progression and altered p107 and p130 levels were observed in suprabasal keratinocytes after pRb knockdown. In contrast, pRb interference in Akata BX1 Burkitt lymphoma cells showed a distinct phenotype from differentiated epithelia with no significant effect on EBV IE or EA-D expression. Instead, pRb knockdown reduced the levels of the plasmablast differentiation marker PRDM1/Blimp1 and increased the abundance of c-Myc protein in reactivated Akata BL with pRb knockdown. c-Myc RNA levels also increased following the loss of pRb in epithelial rafts. These results suggest that pRb is required to suppress c-Myc for efficient EBV replication in BL cells and identifies a mechanism for how HPV immortalization, through degradation of the retinoblastoma pocket proteins, interferes with EBV replication in coinfected epithelia. IMPORTANCE Terminally differentiated epithelium is known to support EBV genome amplification and virion morphogenesis following infection. The contribution of the cell cycle in differentiated tissues to efficient EBV replication is not understood. Using organotypic epithelial raft cultures and genetic interference, we can identify factors required for EBV replication in quiescent cells. Here, we phenocopied HPV16 E7 inhibition of EBV replication through knockdown of pRb. Loss of pRb was found to reduce EBV early gene expression and viral replication. Interruption of the viral life cycle was accompanied by increased S-phase gene expression in postmitotic keratinocytes, a process also observed in E7-positive epithelia, and deregulation of other pocket proteins. Together, these findings provide evidence of a global requirement for pRb in EBV lytic replication and provide a mechanistic framework for how HPV E7 may facilitate a latent EBV infection through its mediated degradation of pRb in copositive epithelia.
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Linfoma de Burkitt , Infecciones por Virus de Epstein-Barr , Proteína de Retinoblastoma , Replicación Viral , Humanos , Linfoma de Burkitt/virología , Diferenciación Celular , Epitelio/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Infecciones por Papillomavirus , Proteína de Retinoblastoma/metabolismoRESUMEN
El virus de Epstein-Barr (VEB) fue el primer virus asociado a neoplasias en humanos. Infecta el 95 % de la población mundial, y aunque usualmente es asintomático, puede causar mononucleosis infecciosa y se relaciona con más de 200.000 casos de neoplasias al año. De igual forma, se asocia con esclerosis múltiple y otras enfermedades autoinmunes. A pesar de ser catalogado como un virus oncogénico, solo un pequeño porcentaje de los individuos infectados desarrollan neoplasias asociadas a VEB. Su persistencia involucra la capacidad de alternar entre una serie de programas de latencia, y de reactivarse cuando tiene la necesidad de colonizar nuevas células B de memoria, con el fin de sostener una infección de por vida y poder transmitirse a nuevos hospederos. En esta revisión se presentan las generalidades del VEB, además de su asociación con varios tipos de neoplasias, como son el carcinoma nasofaríngeo, el carcinoma gástrico, el linfoma de Hodgkin y el linfoma de Burkitt, y la esclerosis múltiple. Adicionalmente, se describen los mecanismos fisiopatológicos de las diferentes entidades, algunos de ellos no completamente dilucidados
Epstein-Barr virus (EBV) was the first virus associated with human cancer. It infects 95% of the world's population, and although it is usually asymptomatic, it causes infectious mononucleosis. It is related to more than 200,000 cases of cancer per year, and is also associated with multiple sclerosis and other autoimmune diseases. Despite being classified as an oncogenic virus, only a small percentage of infected individuals develop EBV-associated cancer. Its persistence involves the ability to alternate between a series of latency programs, and the ability to reactivate itself when it needs to colonize new memory B cells, in order to sustain a lifelong infection and be able to transmit to new hosts. In this review, the general characteristics of EBV are presented, in addition to its association with various types of cancers, such as nasopharyngeal carcinoma, gastric carcinoma, Hodgkin's lymphoma and Burkitt's lymphoma, and multiple sclerosis. Additionally, the pathophysiological mechanisms of the different entities are described, some of them not completely elucidated yet
Asunto(s)
Humanos , Herpesvirus Humano 4/fisiología , Infecciones por Virus de Epstein-Barr/complicaciones , Neoplasias Gástricas/fisiopatología , Neoplasias Gástricas/virología , Enfermedad de Hodgkin/fisiopatología , Enfermedad de Hodgkin/virología , Neoplasias Nasofaríngeas/fisiopatología , Neoplasias Nasofaríngeas/virología , Linfoma de Burkitt/fisiopatología , Linfoma de Burkitt/virología , Carcinogénesis , Carcinoma Nasofaríngeo/fisiopatología , Carcinoma Nasofaríngeo/virología , Esclerosis Múltiple/fisiopatología , Esclerosis Múltiple/virologíaRESUMEN
It is emerging that targeting the adaptive functions of Unfolded Protein Response (UPR) may represent a promising anti-cancer therapeutic approach. This is particularly relevant for B-cell lymphomas, characterized by a high level of constitutive stress due to high c-Myc expression. In this study, we found that IRE1α/XBP1 axis inhibition exerted a stronger cytotoxic effect compared to the inhibition of the other two UPR sensors, namely PERK and ATF6, in Burkitt lymphoma (BL) cells, in correlation with c-Myc downregulation. Interestingly, such an effect was more evident in Epstein-Barr virus (EBV)-negative BL cells or those cells expressing type I latency compared to type III latency BL cells. The other interesting finding of this study was that the inhibition of IRE1α/XBP1 downregulated BRCA-1 and RAD51 and potentiated the cytotoxicity of PARP inhibitor AZD2661 against BL cells and also against Primary Effusion Lymphoma (PEL), another aggressive B-cell lymphoma driven by c-Myc and associated with gammaherpesvirus infection. These results suggest that combining the inhibition of UPR sensors, particularly IRE1α/XBP1 axis, and molecules involved in DDR, such as PARP, could offer a new therapeutic opportunity for treating aggressive B-cell lymphomas such as BL and PEL.
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Linfoma de Burkitt , Infecciones por Virus de Epstein-Barr , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Respuesta de Proteína Desplegada , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/virología , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Herpesvirus Humano 4/fisiología , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
MYC translocations in association with Epstein-Barr virus (EBV) infection are often observed in B-cell lymphomas. A subset of Burkitt lymphoma (BL) expresses EBV latent membrane proteins 1 and 2A (LMP1 and LMP2A) in addition to the typical restricted EBV latent gene expression. EBV-associated diffuse large B-cell lymphoma (DLBCL) typically exhibits latency type II or III and expresses LMP1. Here, we investigate the role of LMP1 in MYC-driven lymphomagenesis in our murine model. λ-MYC mice develop tumors having a "starry sky" appearance and have abnormal p53 expression that is also observed in human BL. LMP2A/λ-MYC double-transgenic mice develop tumors significantly faster than mice only expressing MYC. Similar to LMP2A/λ-MYC mice, LMP1/λ-MYC mice also have accelerated MYC-driven lymphomagenesis. As observed in LMP2A/λ-MYC mice, p27kip1 was degraded in LMP1/λ-MYC pretumor and tumor B cells. Coexpression of LMP1 and LMP2A resulted in the enhancement of B cell proliferation. In contrast to LMP2A, the inhibition of Syk or cyclin-dependant kinase (CDK)4/6 activity did not effectively inhibit LMP1-mediated MYC lymphomagenesis. Also, in contrast to LMP2A, LMP1 did not lessen abnormal p53 expression in λ-MYC tumors. To investigate the significance of LMP1 expression in human BL development, we reanalyzed RNA sequencing (RNA-Seq) data of primary human BL from previous studies. Interestingly, p53 mutations were less observed in LMP1-expressing BL, although they were not significantly changed by EBV infection, indicating LMP1 may lessen p53 mutations in human primary BL. This suggests that LMP1 effects in EBV-associated human BL vary from what we observe in our murine model. Finally, our studies suggest a novel pathogenic role of LMP1 in lymphomagenesis.
Asunto(s)
Linfoma de Burkitt , Infecciones por Virus de Epstein-Barr , Linfoma de Células B Grandes Difuso , Proteínas Proto-Oncogénicas c-myc , Proteínas de la Matriz Viral , Animales , Linfoma de Burkitt/genética , Linfoma de Burkitt/virología , Modelos Animales de Enfermedad , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Humanos , Linfoma de Células B Grandes Difuso/etiología , Linfoma de Células B Grandes Difuso/virología , Ratones , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de la Matriz Viral/metabolismoRESUMEN
Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.
Asunto(s)
Linfocitos B , Linfoma de Burkitt , Herpesvirus Humano 4 , Factores Reguladores del Interferón , Linfocitos B/metabolismo , Linfocitos B/patología , Linfocitos B/virología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Herpesvirus Humano 4/metabolismo , Humanos , Factores Reguladores del Interferón/metabolismo , Fenotipo , Proteínas Virales/metabolismoRESUMEN
SignificanceEpstein-Barr virus (EBV) contributes to Burkitt lymphoma and post-transplant lymphoproliferative disease (PTLD). EBV-transforming programs activate lipid metabolism to convert B cells into immortalized lymphoblastoid cell lines (LCL), a PTLD model. We found that stages of EBV transformation generate lipid reactive oxygen species (ROS) byproducts to varying degrees, and that a Burkitt-like phase of B cell outgrowth requires lipid ROS detoxification by glutathione peroxidase 4 and its cofactor glutathione. Perturbation of this redox defense in early stages of transformation or in Burkitt cells triggered ferroptosis, a programmed cell death pathway. LCLs were less dependent on this defense, a distinction tied to EBV latency programs. This highlights ferroptosis induction as a potential therapeutic approach for prevention or treatment of certain EBV+ lymphomas.
Asunto(s)
Linfocitos B , Linfoma de Burkitt , Transformación Celular Viral , Ferroptosis , Herpesvirus Humano 4 , Latencia del Virus , Linfocitos B/inmunología , Linfocitos B/virología , Linfoma de Burkitt/virología , Ferroptosis/inmunología , Herpesvirus Humano 4/fisiología , Humanos , Metabolismo de los Lípidos , Activación de Linfocitos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Epstein-Barr virus (EBV) is an oncogenic virus found in about 95% of endemic Burkitt lymphoma (BL) cases. In latently infected cells, EBV DNA is mostly maintained in episomal form, but it can also be integrated into the host genome, or both forms can coexist in the infected cells. METHODS: In this study, we mapped the chromosomal integration sites of EBV (EBV-IS) into the genome of 21 EBV+ BL cell lines (BL-CL) using metaphase fluorescence in situ hybridization (FISH). The data were used to investigate the EBV-IS distribution pattern in BL-CL, its relation to the genome instability, and to assess its association to common fragile sites and episomes. RESULTS: We detected a total of 459 EBV-IS integrated into multiple genome localizations with a preference for gene-poor chromosomes. We did not observe any preferential affinity of EBV to integrate into common and rare fragile sites or enrichment of EBV-IS at the chromosomal breakpoints of the BL-CL analyzed here, as other DNA viruses do. CONCLUSIONS: We identified a non-random integration pattern into 13 cytobands, of which eight overlap with the EBV-IS in EBV-transformed lymphoblastoid cell lines and with a preference for gene- and CpGs-poor G-positive cytobands. Moreover, it has been demonstrated that the episomal form of EBV interacts in a non-random manner with gene-poor and AT-rich regions in EBV+ cell lines, which may explain the observed affinity for G-positive cytobands in the EBV integration process. Our results provide new insights into the patterns of EBV integration in BL-CL at the chromosomal level, revealing an unexpected connection between the episomal and integrated forms of EBV.
Asunto(s)
Linfoma de Burkitt/virología , Cromosomas Humanos/genética , Herpesvirus Humano 4/genética , Integración Viral , Secuencia de Bases , Linfoma de Burkitt/genética , Línea Celular Tumoral , ADN Viral/genética , Humanos , PlásmidosRESUMEN
High mobility group box 1 (HMGB1) is an important chromatin protein and a pro-inflammatory molecule. Though shown to enhance target DNA binding by the Epstein-Barr virus (EBV) lytic switch protein ZEBRA, whether HMGB1 actually contributes to gammaherpesvirus biology is not known. In investigating the contribution of HMGB1 to the lytic phase of EBV, important for development of EBV-mediated diseases, we find that compared to latently-infected cells, lytic phase Burkitt lymphoma-derived cells and peripheral blood lytic cells during primary EBV infection express high levels of HMGB1. Our experiments place HMGB1 upstream of ZEBRA and reveal that HMGB1, through the NLRP3 inflammasome, sustains the expression of ZEBRA. These findings indicate that in addition to the NLRP3 inflammasome's recently discovered role in turning the EBV lytic switch on, NLRP3 cooperates with the danger molecule HMGB1 to also maintain ZEBRA expression, thereby sustaining the lytic signal.
Asunto(s)
Linfoma de Burkitt/genética , Infecciones por Virus de Epstein-Barr/genética , Proteína HMGB1/genética , Herpesvirus Humano 4/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Transactivadores/genética , Linfocitos B/inmunología , Linfocitos B/virología , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Regulación Neoplásica de la Expresión Génica , Proteína HMGB1/inmunología , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/patogenicidad , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inflamasomas/genética , Inflamasomas/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Cultivo Primario de Células , Transducción de Señal , Transactivadores/inmunología , Activación Viral/genética , Activación Viral/inmunología , Latencia del Virus/genética , Latencia del Virus/inmunologíaRESUMEN
BACKGROUND: More than 90% of the adult population globally is chronically infected by the Epstein-Barr virus (EBV). It is well established that EBV is associated with a number of malignancies, and advances in knowledge of EBV-related malignancies are being made every year. Several studies have analysed the global epidemiology and geographic distribution of EBV-related cancers. However, most have only described a single cancer type or subtype in isolation or limited their study to the three or four most common EBV-related cancers. This review will present an overview on the spectrum of cancers linked to EBV based on observations of associations and proportions in the published literature while also using these observations to estimate the incidence and mortality burden of some of these cancers. METHOD: We have reviewed the literature on defining features, distribution and outcomes across six cancers with a relatively large EBV-related case burden: Nasopharyngeal carcinoma (NPC), Gastric carcinoma (GC), Hodgkin lymphoma (HL), Burkitt lymphoma (BL), Diffuse large B-cell lymphoma (DLBCL) and Extranodal NK/T-cell lymphoma, Nasal type (ENKTL-NT). We retrieved published region-specific EBV-related case proportions for NPC, GC, HL and BL and performed meta-analyses on pooled region-specific studies of EBV-related case proportions for DLBCL and ENKTL-NT. We match these pooled proportions with their respective regional incidence and mortality numbers retrieved from a publicly available cancer database. Additionally, we also reviewed the literature on several other less common EBV-related cancers to summarize their key characteristics herein. CONCLUSION: We estimated that EBV-related cases from these six cancers accounted for 239,700-357,900 new cases and 137,900-208,700 deaths in 2020. This review highlights the significant global impact of EBV-related cancers and extends the spectrum of disease that could benefit from an EBV-specific therapeutic.
Asunto(s)
Infecciones por Virus de Epstein-Barr/epidemiología , Infecciones por Virus de Epstein-Barr/patología , Herpesvirus Humano 4/patogenicidad , Neoplasias/epidemiología , Neoplasias/virología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bleomicina/uso terapéutico , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/virología , Dacarbazina/uso terapéutico , Doxorrubicina/uso terapéutico , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/virología , Humanos , Linfoma Extranodal de Células NK-T/tratamiento farmacológico , Linfoma Extranodal de Células NK-T/virología , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/virología , Carcinoma Nasofaríngeo/tratamiento farmacológico , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/virología , Neoplasias/tratamiento farmacológico , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/virología , Vinblastina/uso terapéuticoRESUMEN
Epstein-Barr virus (EBV), a human herpesvirus, is several human lymphoid malignancies-associated. Our earlier study found the effect of Polygonum cuspidatum root on promoting EBV-positive apoptosis. Therefore, this study investigated the effects of the Polygonum cuspidatum ethyl acetate subfraction containing emodin on EBV gene expression and anti-EBV tumor cells. Resultantly, the the Polygonum cuspidatum ethyl acetate subfraction containing emodin (F3a) promoted Raji cell death (50% cytotoxic concentration, CC50: 12.08 µg/mL); the 12.5 µg/mL F3a effect transcribed BRLF1 and BNLF1 and increased latent membrane protein 1 (LMP1), which may reduce the intracellular phospho-extracellular signal-regulated kinase (ERK) and phospho-inhibitor of Nuclear factor kappa B α (IκBα). Meanwhile, the Raji cells increased the intracellular reactive-oxygen species (ROS), activated the apoptosis-related proteins, cleaved caspase 3 and poly(ADP-ribose)polymerase (PARP), and increased the apoptosis percentage. Therefore, the Polygonum cuspidatum ethyl acetate subfraction containing emodin could be a therapeutic drug for EBV-related tumors.
Asunto(s)
Emodina/farmacología , Infecciones por Virus de Epstein-Barr/metabolismo , Fallopia japonica/química , Herpesvirus Humano 4/metabolismo , Neoplasias/virología , Extractos Vegetales/farmacología , Proteínas Virales/metabolismo , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis , Linfoma de Burkitt/virología , Línea Celular Tumoral , Emodina/uso terapéutico , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/virología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica , Humanos , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Fitoterapia , Extractos Vegetales/uso terapéutico , Raíces de Plantas/química , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteínas de la Matriz Viral/metabolismoRESUMEN
Viruses and viral components have been shown to manipulate the expression of host microRNAs (miRNAs) to their advantage, and in some cases to play essential roles in cancer pathogenesis. Burkitt lymphoma (BL), a highly aggressive B-cell derived cancer, is significantly over-represented among people infected with HIV. This study adds to accumulating evidence demonstrating that the virus plays a direct role in promoting oncogenesis. A custom miRNA PCR was used to identify 32 miRNAs that were differently expressed in Burkitt lymphoma cells exposed to HIV-1, with a majority of these being associated with oncogenic processes. Of those, hsa-miR-200c-3p, a miRNA that plays a crucial role in cancer cell migration, was found to be significantly downregulated in both the array and in single-tube validation assays. Using an in vitro transwell system we found that this downregulation correlated with significantly enhanced migration of BL cells exposed to HIV-1. Furthermore, the expression of the ZEB1 and ZEB2 transcription factors, which are promotors of tumour invasion and metastasis, and which are direct targets of hsa-miR-200c-3p, were found to be enhanced in these cells. This study therefore identifies novel miRNAs as role players in the development of HIV-associated BL, with one of these miRNAs, hsa-miR-200c-3p, being a candidate for further clinical studies as a potential biomarker for prognosis in patients with Burkitt lymphoma, who are HIV positive.
Asunto(s)
Linfoma de Burkitt/genética , Carcinogénesis/genética , MicroARNs/genética , Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Línea Celular Tumoral , Movimiento Celular , VIH-1/patogenicidad , Humanos , MicroARNs/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Lymphomas remain a leading cause of morbidity and mortality for HIV-positive patients. The most common lymphomas include diffuse large B-cell lymphoma, Burkitt lymphoma, primary effusion lymphoma, plasmablastic lymphoma and Hodgkin lymphoma. Appropriate approach is determined by lymphoma stage, performans status, comorbidities, histological subtype, status of the HIV disease and immunosuppression. Treatment outcomes have improved due to chemotherapy modalities and effective antiretroviral therapy. This review summarizes epidemiology, pathogenesis, pathology, and current treatment landscape in HIV associated lymphoma.