RESUMO
The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.
Assuntos
Memória Imunológica/fisiologia , Linfoma Difuso de Grandes Células B/patologia , Proteínas Nucleares/genética , Células Precursoras de Linfócitos B/imunologia , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Cromatina/química , Cromatina/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Histona Desacetilases/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Correpressor 2 de Receptor Nuclear/química , Correpressor 2 de Receptor Nuclear/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
During the germinal center (GC) reaction, B cells undergo extensive redistribution of cohesin complex and three-dimensional reorganization of their genomes. Yet, the significance of cohesin and architectural programming in the humoral immune response is unknown. Herein we report that homozygous deletion of Smc3, encoding the cohesin ATPase subunit, abrogated GC formation, while, in marked contrast, Smc3 haploinsufficiency resulted in GC hyperplasia, skewing of GC polarity and impaired plasma cell (PC) differentiation. Genome-wide chromosomal conformation and transcriptional profiling revealed defects in GC B cell terminal differentiation programs controlled by the lymphoma epigenetic tumor suppressors Tet2 and Kmt2d and failure of Smc3-haploinsufficient GC B cells to switch from B cell- to PC-defining transcription factors. Smc3 haploinsufficiency preferentially impaired the connectivity of enhancer elements controlling various lymphoma tumor suppressor genes, and, accordingly, Smc3 haploinsufficiency accelerated lymphomagenesis in mice with constitutive Bcl6 expression. Collectively, our data indicate a dose-dependent function for cohesin in humoral immunity to facilitate the B cell to PC phenotypic switch while restricting malignant transformation.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Dosagem de Genes , Centro Germinativo/metabolismo , Imunidade Humoral , Linfoma de Células B/genética , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/deficiência , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Centro Germinativo/imunologia , Centro Germinativo/patologia , Haploinsuficiência , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , CoesinasRESUMO
Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.
Assuntos
Linfócitos B/fisiologia , Biomarcadores Tumorais/genética , Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Linfoma Difuso de Grandes Células B/genética , Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação Neoplásica da Expressão Gênica , Testes Genéticos , Estudo de Associação Genômica Ampla , Antígenos HLA/metabolismo , Humanos , Vigilância Imunológica , Linfoma Difuso de Grandes Células B/metabolismo , Evasão Tumoral/genéticaRESUMO
Research over the past decade has suggested important roles for pseudogenes in physiology and disease. In vitro experiments demonstrated that pseudogenes contribute to cell transformation through several mechanisms. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. Here, we report that mice engineered to overexpress either the full-length murine B-Raf pseudogene Braf-rs1 or its pseudo "CDS" or "3' UTR" develop an aggressive malignancy resembling human diffuse large B cell lymphoma. We show that Braf-rs1 and its human ortholog, BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo. Notably, we find that transcriptional or genomic aberrations of BRAFP1 occur frequently in multiple human cancers, including B cell lymphomas. Our engineered mouse models demonstrate the oncogenic potential of pseudogenes and indicate that ceRNA-mediated microRNA sequestration may contribute to the development of cancer.
Assuntos
Linfoma Difuso de Grandes Células B/genética , Proteínas Proto-Oncogênicas B-raf/genética , Pseudogenes , RNA/metabolismo , Animais , Sequência de Bases , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas B-raf/metabolismoRESUMO
Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting in vivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Quadruplex G , Síndrome de Imunodeficiência com Hiper-IgM/etiologia , Síndrome de Imunodeficiência com Hiper-IgM/metabolismo , Mutação , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biologia Computacional/métodos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Ativação Enzimática , Imunofluorescência , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Síndrome de Imunodeficiência com Hiper-IgM/diagnóstico , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunofenotipagem , Ativação Linfocitária/genética , Linfoma Difuso de Grandes Células B/etiologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos TransgênicosRESUMO
Genes that drive the proliferation, survival, invasion and metastasis of malignant cells have been identified for many human cancers1-4. Independent studies have identified cell death pathways that eliminate cells for the good of the organism5,6. The coexistence of cell death pathways with driver mutations suggests that the cancer driver could be rewired to activate cell death using chemical inducers of proximity (CIPs). Here we describe a new class of molecules called transcriptional/epigenetic CIPs (TCIPs) that recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes, thereby activating their expression. We focused on diffuse large B cell lymphoma, in which the transcription factor B cell lymphoma 6 (BCL6) is deregulated7. BCL6 binds to the promoters of cell death genes and epigenetically suppresses their expression8. We produced TCIPs by covalently linking small molecules that bind BCL6 to those that bind to transcriptional activators that contribute to the oncogenic program, such as BRD4. The most potent molecule, TCIP1, increases binding of BRD4 by 50% over genomic BCL6-binding sites to produce transcriptional elongation at pro-apoptotic target genes within 15 min, while reducing binding of BRD4 over enhancers by only 10%, reflecting a gain-of-function mechanism. TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50 of 1-10 nM in 72 h and exhibits cell-specific and tissue-specific effects, capturing the combinatorial specificity inherent to transcription. The TCIP concept also has therapeutic applications in regulating the expression of genes for regenerative medicine and developmental disorders.
Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B , Fatores de Transcrição , Humanos , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Ciclo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Fatores de Transcrição/metabolismo , Epigênese Genética/efeitos dos fármacos , Regiões Promotoras Genéticas , Carcinogênese/efeitos dos fármacos , Carcinogênese/genéticaRESUMO
Diffuse large B cell lymphoma (DLBCL) is the most common B cell non-Hodgkin lymphoma and remains incurable in around 40% of patients. Efforts to sequence the coding genome identified several genes and pathways that are altered in this disease, including potential therapeutic targets1-5. However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers are highly and specifically hypermutated in 92% of samples from individuals with DLBCL, display signatures of activation-induced cytidine deaminase activity, and are linked to genes that encode B cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated super-enhancers linked to the BCL6, BCL2 and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (targeting BCL6) and the steroid receptor NR3C1 (targeting BCL2 and CXCR4). Genetic correction of selected mutations restored repressor DNA binding, downregulated target gene expression and led to the counter-selection of cells containing corrected alleles, indicating an oncogenic dependency on the super-enhancer mutations. This pervasive super-enhancer mutational mechanism reveals a major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance.
Assuntos
Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B , Mutação , Oncogenes , Regulação para Baixo , Elementos Facilitadores Genéticos/genética , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Oncogenes/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Receptores CXCR4/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Repressoras/metabolismoRESUMO
Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3, a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.
Assuntos
Linfoma Difuso de Grandes Células B , NF-kappa B , Transdução de Sinais , Fator 3 Associado a Receptor de TNF , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Humanos , NF-kappa B/metabolismo , Quinase Induzida por NF-kappaB , Linhagem Celular Tumoral , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proliferação de CélulasRESUMO
ABSTRACT: Hematological malignancies such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B-cell lymphoma (DLBCL) cause significant morbidity in humans. A substantial number of these lymphomas, particularly HL and DLBCLs have poorer prognosis because of their association with Epstein-Barr virus (EBV). Our earlier studies have shown that EBV-encoded nuclear antigen (EBNA2) upregulates programmed cell death ligand 1 in DLBCL and BLs by downregulating microRNA-34a. Here, we investigated whether EBNA2 affects the inducible costimulator (ICOS) ligand (ICOSL), a molecule required for efficient recognition of tumor cells by T cells through the engagement of ICOS on the latter. In virus-infected and EBNA2-transfected B-lymphoma cells, ICOSL expression was reduced. Our investigation of the molecular mechanisms revealed that this was due to an increase in microRNA-24 (miR-24) by EBNA2. By using ICOSL 3' untranslated region-luciferase reporter system, we validated that ICOSL is an authentic miR-24 target. Transfection of anti-miR-24 molecules in EBNA2-expressing lymphoma cells reconstituted ICOSL expression and increased tumor immunogenicity in mixed lymphocyte reactions. Because miR-24 is known to target c-MYC, an oncoprotein positively regulated by EBNA2, we analyzed its expression in anti-miR-24 transfected lymphoma cells. Indeed, the reduction of miR-24 in EBNA2-expressing DLBCL further elevated c-MYC and increased apoptosis. Consistent with the in vitro data, EBNA2-positive DLBCL biopsies expressed low ICOSL and high miR-24. We suggest that EBV evades host immune responses through EBNA2 by inducing miR-24 to reduce ICOSL expression, and for simultaneous rheostatic maintenance of proproliferative c-MYC levels. Overall, these data identify miR-24 as a potential therapeutically relevant target in EBV-associated lymphomas.
Assuntos
Infecções por Vírus Epstein-Barr , Doença de Hodgkin , Linfoma Difuso de Grandes Células B , MicroRNAs , Humanos , Antagomirs , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Doença de Hodgkin/complicações , Ligantes , Linfoma Difuso de Grandes Células B/metabolismo , MicroRNAs/genética , Proteínas Virais/metabolismoRESUMO
Long non-coding RNAs (LncRNAs) could regulate chemoresistance through sponging microRNAs (miRNAs) and sequestering RNA binding proteins. However, the mechanism of lncRNAs in rituximab resistance in diffuse large B-cell lymphoma (DLBCL) is largely unknown. Here, we investigated the functions and molecular mechanisms of lncRNA CHROMR in DLBCL tumorigenesis and chemoresistance. LncRNA CHROMR is highly expressed in DLBCL tissues and cells. We examined the oncogenic functions of lncRNA CHROMR in DLBCL by a panel of gain-or-loss-of-function assays and in vitro experiments. LncRNA CHROMR suppression promotes CD20 transcription in DLBCL cells and inhibits rituximab resistance. RNA immunoprecipitation, RNA pull-down, and dual luciferase reporter assay reveal that lncRNA CHROMR sponges with miR-27b-3p to regulate mesenchymal-epithelial transition factor (MET) levels and Akt signaling in DLBCL cells. Targeting the lncRNA CHROMR/miR-27b-3p/MET axis reduces DLBCL tumorigenesis. Altogether, these findings provide a new regulatory model, lncRNA CHROMR/miR-27b-3p/MET, which can serve as a potential therapeutic target for DLBCL.
Assuntos
Antineoplásicos Imunológicos , Carcinogênese , Resistencia a Medicamentos Antineoplásicos , Linfoma Difuso de Grandes Células B , MicroRNAs , Proteínas Proto-Oncogênicas c-met , RNA Longo não Codificante , Rituximab , Humanos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Rituximab/farmacologia , Rituximab/uso terapêutico , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
The protease activity of the paracaspase MALT1 is central to lymphocyte activation and lymphomagenesis, but how this activity is controlled remains unknown. Here we identify a monoubiquitination of MALT1 on Lys644 that activated the protease function of MALT1. Monoubiquitinated MALT1 had enhanced protease activity, whereas a ubiquitination-deficient MALT1 mutant with replacement of that lysine with arginine (MALT1(K644R)) had less protease activity, which correlated with impaired induction of interleukin 2 (IL-2) via the T cell antigen receptor in activated T cells. Expression of MALT1(K644R) diminished the survival of cells derived from diffuse large B cell lymphoma of the activated B cell-like subtype (ABC DLBCL), which require constitutive protease activity of MALT1 for survival. Thus, monoubiquitination of MALT1 is essential for its catalytic activation and is therefore a potential target for the treatment of ABC-DLBCL and for immunomodulation.
Assuntos
Caspases/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitinação , Substituição de Aminoácidos , Caspases/química , Caspases/genética , Linhagem Celular Tumoral , Ativação Enzimática , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Mutação , NF-kappa B/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Peptídeo Hidrolases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Linfócitos T/metabolismo , Ubiquitina/metabolismoRESUMO
MEF2B encodes a transcriptional activator and is mutated in â¼11% of diffuse large B cell lymphomas (DLBCLs) and â¼12% of follicular lymphomas (FLs). Here we found that MEF2B directly activated the transcription of the proto-oncogene BCL6 in normal germinal-center (GC) B cells and was required for DLBCL proliferation. Mutation of MEF2B resulted in enhanced transcriptional activity of MEF2B either through disruption of its interaction with the corepressor CABIN1 or by rendering it insensitive to inhibitory signaling events mediated by phosphorylation and sumoylation. Consequently, the transcriptional activity of Bcl-6 was deregulated in DLBCLs with MEF2B mutations. Thus, somatic mutations of MEF2B may contribute to lymphomagenesis by deregulating BCL6 expression, and MEF2B may represent an alternative target for blocking Bcl-6 activity in DLBCLs.
Assuntos
Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Proteínas de Domínio MADS/genética , Mutação , Fatores de Regulação Miogênica/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Ciclo Celular/genética , Proliferação de Células , Análise por Conglomerados , Biologia Computacional , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Perfilação da Expressão Gênica , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas de Domínio MADS/química , Proteínas de Domínio MADS/metabolismo , Fatores de Transcrição MEF2 , Camundongos , Simulação de Acoplamento Molecular , Fatores de Regulação Miogênica/química , Fatores de Regulação Miogênica/metabolismo , Ligação Proteica , Conformação Proteica , Proto-Oncogene Mas , Sumoilação/genética , Transcrição GênicaRESUMO
Diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin lymphoma, is characterized by an aggressive clinical course. In approximately one-third of patients with DLBCL, first-line multiagent immunochemotherapy fails to produce a durable response. Molecular heterogeneity and apoptosis resistance pose major therapeutic challenges in DLBCL treatment. To circumvent apoptosis resistance, the induction of ferroptosis might represent a promising strategy for lymphoma therapy. In this study, a compound library, targeting epigenetic modulators, was screened to identify ferroptosis-sensitizing drugs. Strikingly, bromodomain and extra-terminal domain (BET) inhibitors sensitized cells of the germinal center B-cell-like (GCB) subtype of DLBCL to ferroptosis induction and the combination of BET inhibitors with ferroptosis inducers, such as dimethyl fumarate or RSL3, synergized in the killing of DLBCL cells in vitro and in vivo. On the molecular level, the BET protein BRD4 was found to be an essential regulator of ferroptosis suppressor protein 1 expression and thus to protect GCB-DLBCL cells from ferroptosis. Collectively, we identified and characterized BRD4 as an important player in ferroptosis suppression in GCB-DLBCL and provide a rationale for the combination of BET inhibitors with ferroptosis-inducing agents as a novel therapeutic approach for DLBCL treatment.
Assuntos
Ferroptose , Linfoma Difuso de Grandes Células B , Humanos , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfócitos B/patologia , Proteínas de Ciclo CelularRESUMO
A strategy combining targeted therapies is effective in B-cell lymphomas (BCL), such as mantle cell lymphoma (MCL), but acquired resistances remain a recurrent issue. In this study, we performed integrative longitudinal genomic and single-cell RNA-sequencing analyses of patients with MCL who were treated with targeted therapies against CD20, BCL2, and Bruton tyrosine kinase (OAsIs trial). We revealed the emergence of subclones with a selective advantage against OAsIs combination in vivo and showed that resistant cells were characterized by B-cell receptor (BCR)-independent overexpression of NF-κB1 target genes, especially owing to CARD11 mutations. Functional studies demonstrated that CARD11 gain of function not only resulted in BCR independence but also directly increased the transcription of the antiapoptotic BCL2A1, leading to resistance against venetoclax and OAsIs combination. Based on the transcriptional profile of OAsIs-resistant subclones, we designed a 16-gene resistance signature that was also predictive for patients with MCL who were treated with conventional chemotherapy, underlying a common escape mechanism. Among druggable strategies to inhibit CARD11-dependent NF-κB1 transduction, we evaluated the selective inhibition of its essential partner MALT1. We demonstrated that MALT1 protease inhibition led to a reduction in the expression of genes involved in OAsIs resistance, including BCL2A1. Consequently, MALT1 inhibition induced synergistic cell death in combination with BCL2 inhibition, irrespective of CARD11 mutational status, both in vitro and in vivo. Taken together, our study identified mechanisms of resistance to targeted therapies and provided a novel strategy to overcome resistance in aggressive BCL. The OAsIs trial was registered at www.clinicaltrials.gov #NCT02558816.
Assuntos
Linfoma Difuso de Grandes Células B , Linfoma de Célula do Manto , Adulto , Humanos , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular Tumoral , Mutação com Ganho de Função , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma de Célula do Manto/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, although disease stratification using in-depth plasma proteomics has not been performed to date. By measuring more than 1000 proteins in the plasma of 147 DLBCL patients using data-independent acquisition mass spectrometry and antibody array, DLBCL patients were classified into four proteomic subtypes (PS-I-IV). Patients with the PS-IV subtype and worst prognosis had increased levels of proteins involved in inflammation, including a high expression of metalloproteinase inhibitor-1 (TIMP-1) that was associated with poor survival across two validation cohorts (n = 180). Notably, the combination of TIMP-1 with the international prognostic index (IPI) identified 64.00% to 88.24% of relapsed and 65.00% to 80.49% of deceased patients in the discovery and two validation cohorts, which represents a 24.00% to 41.67% and 20.00% to 31.70% improvement compared to the IPI score alone, respectively. Taken together, we demonstrate that DLBCL heterogeneity is reflected in the plasma proteome and that TIMP-1, together with the IPI, could improve the prognostic stratification of patients.
Assuntos
Linfoma Difuso de Grandes Células B , Inibidor Tecidual de Metaloproteinase-1 , Humanos , Prognóstico , Proteômica , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Biomarcadores , Estudos RetrospectivosRESUMO
BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) represents a prevalent malignant tumor, with approximately 40% of patients encountering treatment challenges or relapse attributed to rituximab resistance, primarily due to diminished or absent CD20 expression. Our prior research identified PDK4 as a key driver of rituximab resistance through its negative regulation of CD20 expression. Further investigation into PDK4's resistance mechanism and the development of advanced exosome nanoparticle complexes may unveil novel resistance targets and pave the way for innovative, effective treatment modalities for DLBCL. METHODS: We utilized a DLBCL-resistant cell line with high PDK4 expression (SU-DHL-2/R). We infected it with short hairpin RNA (shRNA) lentivirus for RNA sequencing, aiming to identify significantly downregulated mRNA in resistant cells. Techniques including immunofluorescence, immunohistochemistry, and Western blotting were employed to determine PDK4's localization and expression in resistant cells and its regulatory role in phosphorylation of Histone deacetylase 8 (HDAC8). Furthermore, we engineered advanced exosome nanoparticle complexes, aCD20@ExoCTX/siPDK4, through cellular, genetic, and chemical engineering methods. These nanoparticles underwent characterization via Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM), and their cellular uptake was assessed through flow cytometry. We evaluated the nanoparticles' effects on apoptosis in DLBCL-resistant cells and immune cells using CCK-8 assays and flow cytometry. Additionally, their capacity to counteract resistance and exert anti-tumor effects was tested in a resistant DLBCL mouse model. RESULTS: We found that PDK4 initiates HDAC8 activation by phosphorylating the Ser-39 site, suppressing CD20 protein expression through deacetylation. The aCD20@ExoCTX/siPDK4 nanoparticles served as effective intracellular delivery mechanisms for gene therapy and monoclonal antibodies, simultaneously inducing apoptosis in resistant DLBCL cells and triggering immunogenic cell death in tumor cells. This dual action effectively reversed the immunosuppressive tumor microenvironment, showcasing a synergistic therapeutic effect in a subcutaneous mouse tumor resistance model. CONCLUSIONS: This study demonstrates that PDK4 contributes to rituximab resistance in DLBCL by modulating CD20 expression via HDAC8 phosphorylation. The designed exosome nanoparticles effectively overcome this resistance by targeting the PDK4/HDAC8/CD20 pathway, representing a promising approach for drug delivery and treating patients with Rituximab-resistant DLBCL.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Exossomos , Linfoma Difuso de Grandes Células B , Nanopartículas , Rituximab , Humanos , Exossomos/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/terapia , Rituximab/farmacologia , Rituximab/uso terapêutico , Animais , Camundongos , Nanopartículas/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoma characterized by constitutive NF-κB activation, but whether miR-17â¼92 contributes to this activation remains unclear. Herein, we sought to evaluate the role of miR-17â¼92 in the process of NF-κB activation in ABC-DLBCL. We found that the expression of miR-17â¼92 primary transcript was positively correlated with NF-κB activity, miR-17â¼92 activated the NF-κB signaling in ABC-DLBCL, and its over-expression promoted ABC-DLBCL cell growth, accelerated cell G1 to S phase transition and enhanced cell resistance to NF-κB inhibitor. Importantly, miR-17â¼92 promoted NF-κB activation through directly targeting multiple ubiquitin-editing regulators to lead to increase the K63-linked polyubiquitination and decrease the K48-linked polyubiquitination of RIP1 complex in ABC-DLBCL. We further found that miR-17â¼92 selectively activated IκB-α and NF-κB p65 but not NF-κB p52/p100, and high miR-17â¼92 expression was also associated with poorer outcome in ABC-DLBCL patients. Overall, our results showed that miR-17â¼92 selectively activated the canonical NF-κB signaling via targeting ubiquitin-editing regulators to lead to constitutively NF-κB activation and poorer outcome in ABC-DLBCL. These findings uncovered an innovative function of miR-17â¼92 and previously unappreciated regulatory mechanism of NF-κB activation in ABC-DLBCL. Targeting miR-17â¼92 may thus provide a novel bio-therapeutic strategy for ABC-DLBCL patients.
Assuntos
Linfoma Difuso de Grandes Células B , MicroRNAs , NF-kappa B , Ubiquitinação , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linhagem Celular Tumoral , Transdução de Sinais , Masculino , Feminino , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Regulação Neoplásica da Expressão Gênica , Pessoa de Meia-Idade , Proliferação de Células/genética , RNA Longo não CodificanteRESUMO
Ferroptosis has been regarded as a critical event in the process of diffuse large B cell lymphoma (DLBCL). Sentrin-specific protease 1 (SENP1) has emerged as an oncogene in multiple human malignancies. The present work was to investigate the effects of SENP1 on the progression of DLBCL and the possible regulatory mechanism involving ferroptosis. SENP1 expression in DLBCL tissues, parental and cisplatin-resistant DLBCL cells were, respectively, tested by GEPIA database, RT-qPCR, and Western blot. Cell viability was estimated via CCK-8 assay. Flow cytometry analysis estimated cell apoptosis and cycle. Western blot examined the expression of apoptosis-, cell cycle-, and ferroptosis-associated proteins. TBARS assay and BODIPY 581/591 C11 probe measured lipid peroxidation. Related assay kit assessed total iron levels. CCK-8 and flow cytometry evaluated cisplatin resistance. SENP1 expression was raised in DLBCL tissues and cells. SENP1 knockdown reduced cell viability, boosted cell apoptosis, cell cycle arrest, and elevated cisplatin sensitivity in DLBCL. SENP1 depletion drove the ferroptosis of both parental and cisplatin-resistant DLBCL cells and ferroptosis inhibitor Fer-1 reversed the influences of SENP1 inhibition on cell viability, apoptosis, cell cycle, and cisplatin resistance in DLBCL. Anyway, SENP1 absence might facilitate ferroptosis to obstruct the development of DLBCL and cisplatin resistance.
Assuntos
Apoptose , Pontos de Checagem do Ciclo Celular , Cisplatino , Cisteína Endopeptidases , Resistencia a Medicamentos Antineoplásicos , Ferroptose , Linfoma Difuso de Grandes Células B , Humanos , Cisplatino/farmacologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Ferroptose/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular TumoralRESUMO
BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a malignant tumour. Although some standard therapies have been established to improve the cure rate, they remain ineffective for specific individuals. Therefore, it is meaningful to find more novel therapeutic approaches. Macrophage polarisation is extensively involved in the process of tumour development. Recombinant hirudin (rH) affects macrophages and has been researched frequently in clinical trials lately. Our article validated the regulatory role of rH in macrophage polarisation and the mechanism of PAR-1 by collecting clinical samples and subsequently establishing a cellular model to provide a scientifically supported perspective for discovering new therapeutic approaches. METHOD: We assessed the expression of macrophage polarisation markers, cytokines and PAR-1 in clinical samples. We established a cell model by co-culture with THP-1 and OCI-Ly10 cell. We determined the degree of cell polarisation and expression of validation cytokines by flow cytometry, ELISA, and RT-qPCR to confirm the success of the cell model. Subsequently, different doses of rH were added to discover the function of rH on cell polarisation. We confirmed the mechanism of PAR-1 in macrophage polarisation by transfecting si-PAR-1 and pcDNA3.1-PAR-1. RESULTS: We found higher expression of M2 macrophage markers (CD163 + CMAF+) and PAR-1 in 32 DLBCL samples. After inducing monocyte differentiation into M0 macrophages and co-culturing with OCI-Ly10 lymphoma cells, we found a trend of these expressions in the cell model consistent with the clinical samples. Subsequently, we discovered that rH promotes the polarisation of M1 macrophages but inhibits the polarisation of M2 macrophages. We also found that PAR-1 regulates macrophage polarisation, inhibiting cell proliferation, migration, invasion and angiogenic capacity. CONCLUSION: rH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages.
Assuntos
Hirudinas , Linfoma Difuso de Grandes Células B , Macrófagos , Receptor PAR-1 , Proteínas Recombinantes , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/genética , Humanos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Receptor PAR-1/metabolismo , Receptor PAR-1/genética , Hirudinas/farmacologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Polaridade Celular/efeitos dos fármacos , Feminino , Masculino , Citocinas/metabolismo , Pessoa de Meia-Idade , Células THP-1 , IdosoRESUMO
Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly. We demonstrate that Ly-TME cellular and biophysical factors amplify the BCR-MYD88-TLR9 multiprotein supercomplex and induce cooperative signalling pathways in ABC-DLBCL cells, which reduce the efficacy of compounds targeting the BCR pathway members Bruton tyrosine kinase and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). Combinatorial inhibition of multiple aberrant signalling pathways induced higher antitumour efficacy in lymphoid organoids and implanted ABC-DLBCL patient tumours in vivo. Our studies define the complex crosstalk between malignant ABC-DLBCL cells and Ly-TME, and provide rational combinatorial therapies that rescue Ly-TME-mediated attenuation of treatment response to MALT1 inhibitors.