RESUMO
Primary effusion lymphoma (PEL) is a malignant B-cell lymphoma attributable to Kaposi sarcoma-associated herpesvirus (KSHV) infection. PEL is characterized by invasive behavior, showing recurrent effusions in body cavities. The clinical outcome and typical prognosis in patients with PEL are poor and potentially lethal. Clarification of the pathogenesis in PEL is urgently needed in order to develop novel therapies. PEL cells generally lack B-cell surface markers, and we therefore hypothesized that the B-cell transcription factor, PAX5, would be down-regulated in PEL. The expression of PAX5 is detected from the pro-B to the mature B-cell stage and is indispensable for the differentiation of B-cells. PAX5 was silenced in PEL cells via its promoter methylation. Up-regulation of PAX5 induced several genes coding for B-cell surface marker mRNA, but not protein level. PAX5 inhibited cell growth via G1 cell cycle arrest. PAX5 bound to RB and increased its protein expression. RB/E2F-regulated genes were significantly down-regulated in microarray analysis and PCR experiments. To elucidate the in vivo role of PAX5, we examined the restoration of PAX5 in a PEL mouse model. The ascites volume and organ invasions were significantly suppressed by PAX5 restoration. Reduction of PAX5 has played a crucial role in the oncogenesis of PEL, and PAX5 is a tumor suppressor in PEL. Targeting PAX5 could represent a novel therapeutic strategy for patients with PEL.
Assuntos
Pontos de Checagem do Ciclo Celular , Herpesvirus Humano 8 , Linfoma de Efusão Primária , Fator de Transcrição PAX5 , Fator de Transcrição PAX5/metabolismo , Fator de Transcrição PAX5/genética , Linfoma de Efusão Primária/virologia , Linfoma de Efusão Primária/metabolismo , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/patologia , Linfoma de Efusão Primária/etiologia , Animais , Humanos , Herpesvirus Humano 8/genética , Camundongos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Proliferação de Células , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/complicações , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Regiões Promotoras Genéticas , Modelos Animais de DoençasRESUMO
Primary effusion lymphoma (PEL) is a rare and aggressive B-cell lymphoma, against which current therapies usually fail. In the present study, we show that targeting HSPs, such as HSP27, HSP70 and HSP90, could be an efficient strategy to reduce PEL cell survival, as it induces strong DNA damage, which correlated with an impairment of DDR. Moreover, as HSP27, HSP70 and HSP90 cross talk with STAT3, their inhibition results in STAT3 de-phosphorylation and. On the other hand, the inhibition of STAT3 may downregulate these HSPs. These findings suggest that targeting HSPs has important implications in cancer therapy, as it can reduce the release of cytokines by PEL cells, which, besides affecting their own survival, could negatively influence anti-cancer immune response.
Assuntos
Dano ao DNA , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico HSP90 , Linfoma de Efusão Primária , Terapia de Alvo Molecular , Humanos , Apoptose , Linhagem Celular Tumoral , Citocinas , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Linfoma de Efusão Primária/tratamento farmacológico , Linfoma de Efusão Primária/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) causes several malignancies in people living with HIV, including primary effusion lymphoma (PEL). PEL cell lines exhibit oncogene addictions to both viral and cellular genes. Using CRISPR screens, we previously identified cellular oncogene addictions in PEL cell lines, including MCL1. MCL1 is a member of the BCL2 family, which functions to prevent intrinsic apoptosis and has been implicated in several cancers. Despite the overlapping functions of the BCL2 family members, PEL cells are dependent only on MCL1, suggesting that MCL1 may have nonredundant functions. To investigate why PEL cells exhibit selective addiction to MCL1, we inactivated the intrinsic apoptosis pathway by engineering BAX/BAK1 double knockout cells. In this context, PEL cells become resistant to MCL1 knockdown or MCL1 inactivation by the MCL1 inhibitor S63845, indicating that the main function of MCL1 in PEL cells is to prevent BAX/BAK1-mediated apoptosis. The selective requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Ectopic expression of several BCL2 family proteins, as well as the KSHV BCL2 homolog, significantly decreased basal caspase 3/7 activity and buffered against staurosporine-induced apoptosis. Finally, overexpressed BCL2 family members can functionally substitute for MCL1, when it is inhibited by S63845. Together, our data indicate that the expression levels of the BCL2 family likely explain why PEL tumor cells are highly addicted to MCL1. Importantly, our results suggest that caution should be taken when considering MCL1 inhibitors as a monotherapy regimen for PEL because resistance can develop easily. IMPORTANCE Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus. We showed previously that PEL cell lines require the antiapoptotic protein MCL1 for survival but not the other BCL2 family proteins. This selective dependence on MCL1 is unexpected as the BCL2 family functions similarly in preventing intrinsic apoptosis. Recently, new roles for MCL1 not shared with the BCL2 family have emerged. Here, we show that noncanonical functions of MCL1 are unlikely essential. Instead, MCL1 functions mainly to prevent apoptosis. The specific requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Consistent with this model, shifting these expression ratios changes the requirement away from MCL1 and toward the dominant BCL2 family gene. Together, our results indicate that although MCL1 is an attractive chemotherapeutic target to treat PEL, careful consideration must be taken, as resistance to MCL1-specific inhibitors easily develops through BCL2 family overexpression.
Assuntos
Herpesvirus Humano 8 , Linfoma de Efusão Primária , Humanos , Apoptose , Proteína X Associada a bcl-2/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Herpesvirus Humano 8/fisiologia , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/patologia , Linfoma de Efusão Primária/virologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). Here, we review what is known about human gene essentiality in PEL-derived cell lines. We provide an updated list of PEL-specific human gene dependencies, based on the improved definition of core essential genes across human cancer types. The requirements of PEL cell lines for interferon regulatory factor 4 (IRF4), basic leukine zipper ATF-like transcription factor (BATF), G1/S cyclin D2 (CCND2), CASP8 and FADD like apoptosis regulator (CFLAR), MCL1 apoptosis regulator (MCL1), and murine double minute 2 (MDM2) have been confirmed experimentally. KSHV co-opts IRF4 and BATF to drive superenhancer (SE)-mediated expression of IRF4 itself, MYC, and CCND2. IRF4 dependency of SE-mediated gene expression is shared with Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) and human T-cell leukemia virus type 1-transformed adult T-cell leukemia/lymphoma (ATLL) cell lines, as well as several B-cell lymphomas of nonviral etiology. LCLs and ATLL cell lines similarly share dependencies on CCND2 and CFLAR with PEL, but also have distinct gene dependencies. Genetic dependencies could be exploited for therapeutic intervention in PEL and other cancers.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 8 , Leucemia-Linfoma de Células T do Adulto , Linfoma de Efusão Primária , Neoplasias , Adulto , Humanos , Animais , Camundongos , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/tratamento farmacológico , Linfoma de Efusão Primária/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/uso terapêutico , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/genéticaRESUMO
Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors.
Assuntos
Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma de Efusão Primária/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imidazóis/farmacologia , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Linfoma Anaplásico de Células Grandes/genética , Linfoma de Efusão Primária/tratamento farmacológico , Linfoma de Efusão Primária/genética , Compostos Macrocíclicos/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quinolinas/farmacologiaRESUMO
The induction of DNA damage together with the interference with DNA repair represents a promising strategy in cancer treatment. Here we show that the PARP-1/2/3 inhibitor AZD2461 in combination with the CHK1 inhibitor UCN-01 altered the DNA damage response and reduced cell proliferation in PEL cells, an aggressive B cell lymphoma highly resistant to chemotherapies. AZD2461/UCN-01 combination activated p53/p21 and downregulated c-Myc in these cells, leading to a reduced expression level of RAD51, molecule involved in DNA repair. The effect of AZD2461/UCN-01 on c-Myc and p53/p21 was inter-dependent and, besides impairing cell proliferation, contributed to the activation of the replicative cycle of KSHV, carried in a latent state in PEL cells. Finally, we found that the pharmacological or genetic inhibition of p21 counteracted the viral lytic cycle activation and further reduced PEL cell proliferation, suggesting that it could induce a double beneficial effect in this setting. This study unveils that, therapeutic approaches, based on the induction of DNA damage and the reduction of DNA repair, could be used to successfully treat this malignant lymphoma.
Assuntos
Proliferação de Células , Dano ao DNA , Linfoma de Efusão Primária/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Replicação Viral , Linhagem Celular , Células Cultivadas , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/metabolismo , Herpesvirus Humano 8/fisiologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/virologia , Ftalazinas/farmacologia , Piperidinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/toxicidade , Inibidores de Proteínas Quinases/toxicidade , Estaurosporina/análogos & derivados , Estaurosporina/farmacologiaRESUMO
Primary effusion lymphoma (PEL) is an aggressive B cell lymphoma that is etiologically linked to Kaposi's sarcoma-associated herpesvirus (KSHV). Despite standard multi-chemotherapy treatment, PEL continues to cause high mortality. Thus, new strategies to control PEL are needed urgently. Here, we show that a phosphodegron motif within the KSHV protein, latency-associated nuclear antigen (LANA), specifically interacts with E3 ubiquitin ligase FBW7, thereby competitively inhibiting the binding of the anti-apoptotic protein MCL-1 to FBW7. Consequently, LANA-FBW7 interaction enhances the stability of MCL-1 by preventing its proteasome-mediated degradation, which inhibits caspase-3-mediated apoptosis in PEL cells. Importantly, MCL-1 inhibitors markedly suppress colony formation on soft agar and tumor growth of KSHV+PEL/BCBL-1 in a xenograft mouse model. These results strongly support the conclusion that high levels of MCL-1 expression enable the oncogenesis of PEL cells and thus, MCL-1 could be a potential drug target for KSHV-associated PEL. This work also unravels a mechanism by which an oncogenic virus perturbs a key component of the ubiquitination pathway to induce tumorigenesis.
Assuntos
Antígenos Virais/metabolismo , Proteína 7 com Repetições F-Box-WD/metabolismo , Herpesvirus Humano 8/fisiologia , Linfoma de Efusão Primária/virologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Nucleares/metabolismo , Sarcoma de Kaposi/virologia , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Apoptose , Proliferação de Células , Proteína 7 com Repetições F-Box-WD/genética , Feminino , Humanos , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/metabolismo , Linfoma de Efusão Primária/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Nucleares/genética , Fosforilação , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Células Tumorais Cultivadas , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Primary effusion lymphoma (PEL) is a very rare non-Hodgkin lymphoma caused by human herpesvirus-8 (HHV8) that grows in liquid phase within body cavities. The diagnosis of PEL is based on cytology but requires confirmatory ancillary tests. PEL occurs mainly in association with HIV infection. This study describes 9 cases of PEL in HIV-negative patients and compares their characteristics with 10 HIV-associated cases of PEL diagnosed at a single institution in Italy between 1995 and 2019. METHODS: Clinical records were reviewed for demographic data, comorbidities, laboratory abnormalities, and outcome. PEL samples were evaluated for cytomorphology, immunophenotype, immunoglobulin (IG)/T cell receptor (TR) rearrangements, and HHV8 and Epstein-Barr virus (EBV) viral loads in effusion supernatants. RESULTS: HIV-unrelated PEL occurred in 8 elderly patients (7 men, 1 woman) and 1 young adult with primary antibody deficiency. Cytology revealed HHV8-positive lymphoma cells lacking B/T cell antigens and exhibiting 2 cell patterns (polymorphous or monotonous). IG was clonally rearranged in all cases; aberrant TRG occurred in 2 cases. Effusion supernatants had more than 106 HHV8 DNA copies per mL and variable loads of EBV DNA. Compared with HIV-associated PEL, the HIV-negative cohort was characterized by older age, less frequent association with Kaposi sarcoma and/or multicentric Castleman disease, comparable but less abnormal laboratory parameters, and a nonsignificant survival benefit. PEL cases with low apoptosis were associated with better prognosis. CONCLUSION: To the best of our knowledge, our case series of HIV-unrelated PEL is the largest thus far, expands the spectrum of cytological findings, and supports the need for a multidisciplinary approach in the diagnostic workup.
Assuntos
Soronegatividade para HIV , Herpesvirus Humano 8/isolamento & purificação , Linfoma de Efusão Primária/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Genes de Imunoglobulinas , Humanos , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/patologia , Linfoma de Efusão Primária/virologia , Masculino , Pessoa de Meia-IdadeRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is a gamma herpesvirus associated with several human malignancies. Transposable elements (TEs) are ubiquitous in eukaryotic genomes, occupying about 45% of the human genome. TEs have been linked with a variety of disorders and malignancies, though the precise nature of their contribution to many of them has yet to be elucidated. Global transcriptome analysis for differentially expressed TEs in KSHV-associated primary effusion lymphoma (PEL) cells (BCBL1 and BC3) revealed large number of differentially expressed TEs. These differentially expressed TEs include LTR transposons, long interspersed nuclear elements (LINEs), and short interspersed nuclear elements (SINEs). Further analysis of LINE-1 (L1) elements revealed expression upregulation, hypo-methylation, and transition into open chromatin in PEL. In agreement with high L1 expression, PEL cells express ORF1 protein and possess high reverse transcriptase (RT)-activity. Interestingly, inhibition of this RT-activity suppressed PEL cell growth. Collectively, we identified high expression of TEs, and specifically of L1 as a critical component in the proliferation of PEL cells. This observation is relevant for the treatment of KSHV-associated malignancies since they often develop in AIDS patients that are treated with RT inhibitors with potent inhibition for both HIV and L1 RT activity.
Assuntos
Regulação Neoplásica da Expressão Gênica , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Linfoma de Efusão Primária/metabolismo , Linhagem Celular Tumoral , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Herpesvirus Humano 8/genética , Humanos , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/patologia , Linfoma de Efusão Primária/virologiaRESUMO
Primary effusion lymphoma (PEL) has a very poor prognosis. To evaluate the contributions of enhancers/promoters interactions to PEL cell growth and survival, here we produce H3K27ac HiChIP datasets in PEL cells. This allows us to generate the PEL enhancer connectome, which links enhancers and promoters in PEL genome-wide. We identify more than 8000 genomic interactions in each PEL cell line. By incorporating HiChIP data with H3K27ac ChIP-seq data, we identify interactions between enhancers/enhancers, enhancers/promoters, and promoters/promoters. HiChIP further links PEL super-enhancers to PEL dependency factors MYC, IRF4, MCL1, CCND2, MDM2, and CFLAR. CRISPR knock out of MEF2C and IRF4 significantly reduces MYC and IRF4 super-enhancer H3K27ac signal. Knock out also reduces MYC and IRF4 expression. CRISPRi perturbation of these super-enhancers by tethering transcription repressors to enhancers significantly reduces target gene expression and reduces PEL cell growth. These data provide insights into PEL molecular pathogenesis.
Assuntos
Elementos Facilitadores Genéticos/genética , Redes Reguladoras de Genes , Linfoma de Efusão Primária/genética , Regiões Promotoras Genéticas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sequenciamento de Cromatina por Imunoprecipitação , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Herpesvirus Humano 8/patogenicidade , Histonas/genética , Humanos , Fatores Reguladores de Interferon/genética , Linfoma de Efusão Primária/patologia , Linfoma de Efusão Primária/virologia , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The main proliferating component of KS tumors is a cell of endothelial origin termed the spindle cell. Spindle cells are predominantly latently infected with only a small percentage of cells undergoing viral replication. As there is no direct treatment for latent KSHV, identification of host vulnerabilities in latently infected endothelial cells could be exploited to inhibit KSHV-associated tumor cells. Using a pooled CRISPR-Cas9 lentivirus library, we identified host factors that are essential for the survival or proliferation of latently infected endothelial cells in culture, but not their uninfected counterparts. Among the many host genes identified, there was an enrichment in genes localizing to the mitochondria, including genes involved in mitochondrial translation. Antibiotics that inhibit bacterial and mitochondrial translation specifically inhibited the expansion of latently infected endothelial cells and led to increased cell death in patient-derived PEL cell lines. Direct inhibition of mitochondrial respiration or ablation of mitochondrial genomes leads to increased death in latently infected cells. KSHV latent infection decreases mitochondrial numbers, but there are increases in mitochondrial size, genome copy number, and transcript levels. We found that multiple gene products of the latent locus localize to the mitochondria. During latent infection, KSHV significantly alters mitochondrial biology, leading to enhanced sensitivity to inhibition of mitochondrial respiration, which provides a potential therapeutic avenue for KSHV-associated cancers.
Assuntos
Sistemas CRISPR-Cas , Infecções por Herpesviridae/genética , Herpesvirus Humano 8/genética , Mitocôndrias/metabolismo , Latência Viral/genética , Linhagem Celular , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células Endoteliais/metabolismo , Herpesvirus Humano 8/fisiologia , Humanos , Linfoma de Efusão Primária/genética , Sarcoma de Kaposi , Replicação ViralRESUMO
Enhancers play indispensable roles in cell proliferation and survival through spatiotemporally regulating gene transcription. Active enhancers and superenhancers often produce noncoding enhancer RNAs (eRNAs) that precisely control RNA polymerase II activity. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic gamma-2 herpesvirus that causes Kaposi's sarcoma and primary effusion lymphoma (PEL). It is well characterized that KSHV utilizes host epigenetic machineries to control the switch between two lifecycles, latency and lytic replication. However, how KSHV impacts host epigenome at different stages of viral lifecycle is not well understood. Using global run-on sequencing (GRO-seq) and chromatin-immunoprecipitation sequencing (ChIP-seq), we profiled the dynamics of host transcriptional regulatory elements during latency and lytic replication of KSHV-infected PEL cells. This revealed that a number of critical host genes for KSHV latency, including MYC proto-oncogene, were under the control of superenhancers whose activities were globally repressed upon viral reactivation. The eRNA-expressing MYC superenhancers were located downstream of the MYC gene in KSHV-infected PELs and played a key role in MYC expression. RNAi-mediated depletion or dCas9-KRAB CRISPR inhibition of eRNA expression significantly reduced MYC mRNA level in PELs, as did the treatment of an epigenomic drug that globally blocks superenhancer function. Finally, while cellular IRF4 acted upon eRNA expression and superenhancer function for MYC expression during latency, KSHV viral IRF4 repressed cellular IRF4 expression, decreasing MYC expression and thereby, facilitating lytic replication. These results indicate that KSHV acts as an epigenomic driver that modifies host epigenomic status upon reactivation by effectively regulating host enhancer function.
Assuntos
Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 8/genética , Linfoma de Efusão Primária/genética , Linhagem Celular , Epigenômica/métodos , Genes myc/genética , Herpesvirus Humano 8/patogenicidade , Humanos , Proteínas Imediatamente Precoces/genética , Linfoma de Efusão Primária/metabolismo , Linfoma de Efusão Primária/virologia , Proteínas Nucleares/metabolismo , Proto-Oncogene Mas , RNA/metabolismo , Sarcoma de Kaposi/virologia , Transativadores/metabolismo , Transcrição Gênica/genética , Proteínas Virais/metabolismo , Ativação Viral/genética , Latência Viral/genética , Replicação Viral/genéticaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). The cellular transcription factor (TF) interferon (IFN) regulatory factor 4 (IRF4) is an essential oncogene in PEL, but its specific role in PEL and how KSHV deregulates IRF4 remain unknown. Here, we report that the KSHV latency protein viral interferon regulatory factor 3 (vIRF3) cooperates with IRF4 and cellular BATF (basic leucine zipper ATF-like TF) to drive a super-enhancer (SE)-mediated oncogenic transcriptional program in PEL. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) experiments demonstrated that IRF4, vIRF3, and BATF cooccupy the SEs of key survival genes, in a pattern that is distinct from those seen with other IRF4-driven malignancies. All three proteins cooperatively drive SE-mediated IRF4 overexpression. Inactivation of vIRF3 and, to a lesser extent, BATF phenocopies the gene expression changes and loss of cellular viability observed upon inactivation of IRF4. In sum, this work suggests that KSHV vIRF3 and cellular IRF4 and BATF cooperate as oncogenic transcription factors on SEs to promote cellular survival and proliferation in KSHV-associated lymphomas.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes the aggressive disease primary effusion lymphoma (PEL). Here, we show that a viral transcription factor (vIRF3) cooperates with the cellular transcription factor IRF4 to control an oncogenic gene expression program in PEL cells. These proteins promote KSHV-mediated B cell transformation by activating the expression of prosurvival genes through super-enhancers. Our report thus demonstrates that this DNA tumor virus encodes a transcription factor that functions with cellular IRF4 to drive oncogenic transcriptional reprogramming.
Assuntos
Expressão Gênica , Herpesvirus Humano 8/patogenicidade , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/virologia , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Linhagem Celular Tumoral , Humanos , Fatores Reguladores de Interferon/genética , Proteínas Virais/genética , Latência ViralRESUMO
Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved RNA decay mechanism that has emerged as a potent cell-intrinsic restriction mechanism of retroviruses and positive-strand RNA viruses. However, whether NMD is capable of restricting DNA viruses is not known. The DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma and primary effusion lymphoma (PEL). Here, we demonstrate that NMD restricts KSHV lytic reactivation. Leveraging high-throughput transcriptomics we identify NMD targets transcriptome-wide in PEL cells and identify host and viral RNAs as substrates. Moreover, we identified an NMD-regulated link between activation of the unfolded protein response and transcriptional activation of the main KSHV transcription factor RTA, itself an NMD target. Collectively, our study describes an intricate relationship between cellular targets of an RNA quality control pathway and KSHV lytic gene expression, and demonstrates that NMD can function as a cell intrinsic restriction mechanism acting upon DNA viruses.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Viral/metabolismo , Ativação Viral/genética , Linhagem Celular Tumoral , Células HEK293 , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/virologia , RNA Mensageiro/metabolismo , RNA-Seq , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Resposta a Proteínas não Dobradas/genética , Latência Viral/genéticaRESUMO
Protein-protein interactions featuring intricate binding epitopes remain challenging targets for synthetic inhibitors. Interactions of NEMO, a scaffolding protein central to NF-κB signaling, exemplify this challenge. Various regulators are known to interact with different coiled coil regions of NEMO, but the topological complexity of this protein has limited inhibitor design. We undertook a comprehensive effort to block the interaction between vFLIP, a Kaposi's sarcoma herpesviral oncoprotein, and NEMO using small molecule screening and rational design. Our efforts reveal that a tertiary protein structure mimic of NEMO is necessary for potent inhibition. The rationally designed mimic engages vFLIP directly causing complex disruption, protein degradation and suppression of NF-κB signaling in primary effusion lymphoma (PEL). NEMO mimic treatment induces cell death and delays tumor growth in a PEL xenograft model. Our studies with this inhibitor reveal the critical nexus of signaling complex stability in the regulation of NF-κB by a viral oncoprotein.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma de Efusão Primária/metabolismo , NF-kappa B/metabolismo , Animais , Linhagem Celular , Dicroísmo Circular , Herpesvirus Humano 8/metabolismo , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfoma de Efusão Primária/genética , Masculino , Camundongos , Microscopia Confocal , Modelos Biológicos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Primary effusion lymphoma (PEL) is an aggressive neoplasm correlated with human herpesvirus 8 (HHV8). Metabolic reprogramming is a hallmark of cancers. The alterations in cellular metabolism are important to the survival of HHV8 latently infected cells. Pyruvate dehydrogenase (PDH) controls the flux of metabolites between glycolysis and the tricarboxylic acid cycle (TCA cycle) and is a key enzyme in cancer metabolic reprogramming. Glutaminolysis is required for the survival of PEL cells. Glutamate dehydrogenase 1 (GDH1) converts glutamate into α-ketoglutarate supplying the TCA cycle with intermediates to support anaplerosis. Previously we have observed that epigallocatechin-3-gallate (EGCG) can induce PEL cell death and N-acetyl cysteine (NAC) attenuates EGCG induced PEL cell death. In this study, results showed that EGCG upregulated the expression of glucose transporter GLUT3, and reduced the expression of pyruvate dehydrogenase E1-alpha (PDHA1), the major regulator of PDH, and GDH1. NAC could partially reverse the effects of EGCG in PEL cells. Overexpression of PDHA1 in PEL cells or supplement of α-ketoglutarate attenuated EGCG induced cell death. EGCG also reduced the levels of oncometabolite D-2-hydroxyglutarate (D2HG). These results suggest that EGCG may modulate the metabolism of PEL cells leading to cell death.
Assuntos
Catequina/análogos & derivados , Herpesvirus Humano 8 , Linfoma de Efusão Primária/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Piruvato Desidrogenase (Lipoamida)/genética , Catequina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Glutaratos/metabolismo , Humanos , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/virologiaRESUMO
Plasmablastic neoplasms encompass several entities including plasmablastic lymphoma, plasmablastic plasmacytoma/multiple myeloma, primary effusion lymphoma and its extracavitary variant, anaplastic lymphoma kinase-positive large B-cell lymphoma, and Kaposi sarcoma-associated herpesvirus/human herpesvirus 8 (HHV8)-positive diffuse large B-cell lymphoma, not otherwise specified. Morphologically, the tumor cells are large with eccentrically located nuclei, prominent nucleoli, and basophilic/amphophilic cytoplasm. Immunophenotypically, the tumor cells express plasma cell-related antigens including CD38, CD138, interferon regulatory factor-4 (IRF4)/MUM1, PR domain zinc finger protein-1 (PRDM1), and/or X-box binding protein-1 (XBP1), with frequent loss of CD20. These tumors are diagnostically challenging for general pathologists due to their overlapping morphology and immunophenotype, and due to their rarity, and particularly so when clinical and radiologic information is insufficient. We also discuss HHV8-negative effusion-based lymphoma due to its overlapping features with primary effusion lymphoma. In this review, we focus on the useful diagnostic markers and pertinent molecular findings in these distinct entities and propose a practical diagnostic algorithm using anaplastic lymphoma kinase, HHV8, in situ hybridization for Epstein-Barr virus-encoded small RNA, immunoglobulin M, light chain stains, and clinicoradiologic criteria to avoid misdiagnosis. At the molecular level, MYC protein overexpression with or without MYC rearrangement and PRDM1-inactivating mutations or deletions are noted in a subset of such tumors, especially in plasmablastic lymphoma. Prognosis in these entities is dismal with conventional CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy. Therefore, novel target therapies, such as anti-CD30 agents, and/or immune blockade therapy, are potential treatment options in the future.
Assuntos
Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/patologia , Linfoma de Efusão Primária/diagnóstico , Linfoma de Efusão Primária/patologia , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Perfil Genético , Herpesvirus Humano 8 , Imunofenotipagem , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/virologia , Linfoma de Efusão Primária/genéticaRESUMO
Primary effusion lymphoma (PEL) is a rare form of aggressive B-cell lymphoma characterized by a malignant serous effusion involving body cavities. It usually associated with human herpes virus-8 (HHV-8) and coexpression of Epstein-Barr virus and mostly affects patients with HIV. We report a rare case of cutaneous PEL with an unusual intravascular presentation, combined with Kaposi sarcoma involving the skin, lung, and gastrointestinal tract. The molecular genetic analysis of the sarcoma and lymphoma components, using next-generation sequencing was performed. The patient was a 67-year-old man who presented with multiple cutaneous tumors and mass in the left lung. He died 17 hours after the admission to the hospital. At autopsy, in addition to the cutaneous lesions, tumors in the left lung and gastrointestinal mucosa were detected, and no effusions in the body cavities were seen. The biopsy from the cutaneous lesions, pulmonary, and intestinal tumors revealed histological and immunohistochemical features of Kaposi sarcoma. In addition, the skin biopsy specimens contained a diffuse infiltrate composed of large pleomorphic cells, with focal intravascular growth that were negative for pan B-cell markers, weakly positive for CD38 and CD138 but expressed CD3, HHV-8, and Epstein-Barr virus. Molecular genetic studies in this specimen revealed monoclonal rearrangements of the IgH gene. The diagnosis of PEL, solid variant, was made. Next-generation sequencing analysis of the tumorous and normal tissue detected a pathogenic germline mutation of the FAM175A gene and somatic mutations in BRCA2 and RAD51B (in both sarcoma and lymphoma specimens), and INPP4B and RICTOR (in lymphoma specimen only).
Assuntos
Proteínas de Transporte/genética , Linfoma de Efusão Primária/patologia , Sarcoma de Kaposi/patologia , Idoso , Mutação em Linhagem Germinativa , Humanos , Linfoma de Efusão Primária/complicações , Linfoma de Efusão Primária/genética , Masculino , Sarcoma de Kaposi/complicações , Sarcoma de Kaposi/genéticaRESUMO
Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), a gamma-2 herpesvirus, is the causative agent of KS, primary effusion lymphoma (PEL), and a plasma cell variant of multicentric Castleman's disease. Although KSHV latency is detected in KS-related tumors, oncogenic pathways activated by KSHV latent infection are not fully understood. Here, we found that retrotransposition of long interspersed element-1 (L1), a retrotransposon in the human genome, was enhanced in PEL cells. Among the KSHV latent genes, viral FLICE-inhibitory protein (vFLIP) enhanced L1 retrotransposition in an NF-κB-dependent manner. Intracellular cell adhesion molecule-1 (ICAM-1), an NF-κB target, regulated the vFLIP-mediated enhancement of L1 retrotransposition. Furthermore, ICAM-1 downregulated the expression of Moloney leukemia virus 10 (MOV10), an L1 restriction factor. Knockdown of ICAM-1 or overexpression of MOV10 relieved the vFLIP-mediated enhancement of L1 retrotransposition. Collectively, during KSHV latency, vFLIP upregulates ICAM-1 in an NF-κB-dependent manner, which, in turn, downregulates MOV10 expression and thereby enhances L1 retrotransposition. Because active L1 retrotransposition can lead to genomic instability, which is commonly found in KS and PEL, activation of L1 retrotransposition during KSHV latency may accelerate oncogenic processes through enhancing genomic instability. Our results suggest that L1 retrotransposition may be a novel target for impeding tumor development in KSHV-infected patients.