RESUMO
The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukaemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In contrast to EBV- tumours, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL, and they also showed CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal centre DLBCL phenotype, EBV latency programme type II or III, and very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoural B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/- IL2γc-/- mice. Remarkably, the recipients' impaired immunosurveillance favoured the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients samples, but not from healthy donors. Eventually, these cells generated monoclonal tumours (mostly CLL-unrelated but also CLL-related), recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts showed indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and B-cell receptor signalling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Herpesvirus Humano 4/efeitos dos fármacos , Imunossupressores/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Adulto , Idoso , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Transformação Celular Neoplásica/patologia , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/patologia , Feminino , Herpesvirus Humano 4/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Masculino , Pessoa de Meia-IdadeRESUMO
Regulatory T (Treg) cells can weaken antitumor immune responses, and inhibition of their function appears to be a promising therapeutic approach in cancer patients. Mice with targeted deletion of the gene encoding the Cl-/HCO3- anion exchanger AE2 (also termed SLC4A2), a membrane-bound carrier involved in intracellular pH regulation, showed a progressive decrease in the number of Treg cells. We therefore challenged AE2 as a potential target for tumor therapy, and generated linear peptides designed to bind the third extracellular loop of AE2, which is crucial for its exchange activity. Peptide p17AE2 exhibited optimal interaction ability and indeed promoted apoptosis in mouse and human Treg cells, while activating effector T-cell function. Interestingly, this linear peptide also induced apoptosis in different types of human leukemia, lymphoma and multiple myeloma cell lines and primary malignant samples, while it showed only moderate effects on normal B lymphocytes. Finally, a macrocyclic AE2 targeting peptide exhibiting increased stability in vivo was effective in mice xenografted with B-cell lymphoma. These data suggest that targeting the anion exchanger AE2 with specific peptides may represent an effective therapeutic approach in B-cell malignancies.
Assuntos
Antineoplásicos/farmacologia , Antiportadores de Cloreto-Bicarbonato/antagonistas & inibidores , Leucemia de Células B/metabolismo , Linfoma de Células B/metabolismo , Peptídeos/farmacologia , Animais , Ânions/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/patologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas.
Assuntos
Proteínas de Homeodomínio/genética , Linfócitos/metabolismo , Linfoma de Zona Marginal Tipo Células B/genética , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Estimativa de Kaplan-Meier , Tecido Linfoide/metabolismo , Linfoma de Zona Marginal Tipo Células B/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Receptores de Antígenos de Linfócitos B/metabolismo , Quinase Syk/genética , Quinase Syk/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2 and KLF2 genes are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted; therefore, identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here we integrated genome-wide DNA-promoter methylation profiling with gene expression profiling, and clinical and biological variables. An unsupervised clustering analysis of a test series of 98 samples identified 2 clusters with different degrees of promoter methylation. The cluster comprising samples with higher-promoter methylation (High-M) had a poorer overall survival compared with the lower (Low-M) cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2 gene, 7q31-32 loss, and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several prosurvival lymphoma genes were unmethylated and overexpressed. A model based on the methylation of 3 genes (CACNB2, HTRA1, KLF4) identified a poorer-outcome patient subset. Exposure of splenic marginal zone lymphoma cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation.
Assuntos
Metilação de DNA , Linfoma de Zona Marginal Tipo Células B/genética , Neoplasias Esplênicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Transformação Celular Neoplásica , Análise por Conglomerados , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Fator 4 Semelhante a Kruppel , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Prognóstico , Regiões Promotoras Genéticas , Neoplasias Esplênicas/diagnóstico , Neoplasias Esplênicas/mortalidade , Resultado do TratamentoRESUMO
We have previously reported that LITAF is silenced by promoter hypermethylation in germinal centre-derived B-cell lymphomas, but beyond these data the regulation and function of lipopolysaccharide-induced tumour necrosis factor (TNF) factor (LITAF) in B cells are unknown. Gene expression and immunohistochemical studies revealed that LITAF and BCL6 show opposite expression in tonsil B-cell subpopulations and B-cell lymphomas, suggesting that BCL6 may regulate LITAF expression. Accordingly, BCL6 silencing increased LITAF expression, and chromatin immunoprecipitation and luciferase reporter assays demonstrated a direct transcriptional repression of LITAF by BCL6. Gain- and loss-of-function experiments in different B-cell lymphoma cell lines revealed that, in contrast to its function in monocytes, LITAF does not induce lipopolysaccharide-mediated TNF secretion in B cells. However, gene expression microarrays defined a LITAF-related transcriptional signature containing genes regulating autophagy, including MAP1LC3B (LC3B). In addition, immunofluorescence analysis co-localized LITAF with autophagosomes, further suggesting a possible role in autophagy modulation. Accordingly, ectopic LITAF expression in B-cell lymphoma cells enhanced autophagy responses to starvation, which were impaired upon LITAF silencing. Our results indicate that the BCL6-mediated transcriptional repression of LITAF may inhibit autophagy in B cells during the germinal centre reaction, and suggest that the constitutive repression of autophagy responses in BCL6-driven lymphomas may contribute to lymphomagenesis.
Assuntos
Autofagia/genética , Linfoma de Células B/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Subpopulações de Linfócitos B/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Íntrons , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6 , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
B-cell maturation and germinal center (GC) formation are dependent on the interplay between BCL6 and other transcriptional regulators. FOXP1 is a transcription factor that regulates early B-cell development, but whether it plays a role in mature B cells is unknown. Analysis of human tonsillar B-cell subpopulations revealed that FOXP1 shows the opposite expression pattern to BCL6, suggesting that FOXP1 regulates the transition from resting follicular B cell to activated GC B cell. Chromatin immunoprecipitation-on-chip and gene expression assays on B cells indicated that FOXP1 acts as a transcriptional activator and repressor of genes involved in the GC reaction, half of which are also BCL6 targets. To study FOXP1 function in vivo, we developed transgenic mice expressing human FOXP1 in lymphoid cells. These mice exhibited irregular formation of splenic GCs, showing a modest increase in naïve and marginal-zone B cells and a significant decrease in GC B cells. Furthermore, aberrant expression of FOXP1 impaired transcription of noncoding γ1 germline transcripts and inhibited efficient class switching to the immunoglobulin G1 isotype. These studies show that FOXP1 is physiologically downregulated in GC B cells and that aberrant expression of FOXP1 impairs mechanisms triggered by B-cell activation, potentially contributing to B-cell lymphomagenesis.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Centro Germinativo/citologia , Linfoma/imunologia , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular/imunologia , Linhagem Celular , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/imunologia , Fatores de Transcrição Forkhead/imunologia , Centro Germinativo/imunologia , Humanos , Linfoma/metabolismo , Camundongos , Camundongos Transgênicos , Tonsila Palatina/citologia , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Repressoras/imunologia , Ativação Transcricional/imunologiaRESUMO
Earlier work demonstrated that the transcription factor C/EBPα can convert immature and mature murine B lineage cells into functional macrophages. Testing >20 human lymphoma and leukemia B cell lines, we found that most can be transdifferentiated at least partially into macrophage-like cells, provided that C/EBPα is expressed at sufficiently high levels. A tamoxifen-inducible subclone of the Seraphina Burkitt lymphoma line, expressing C/EBPαER, could be efficiently converted into phagocytic and quiescent cells with a transcriptome resembling normal macrophages. The converted cells retained their phenotype even when C/EBPα was inactivated, a hallmark of cell reprogramming. Interestingly, C/EBPα induction also impaired the cells' tumorigenicity. Likewise, C/EBPα efficiently converted a lymphoblastic leukemia B cell line into macrophage-like cells, again dramatically impairing their tumorigenicity. Our experiments show that human cancer cells can be induced by C/EBPα to transdifferentiate into seemingly normal cells at high frequencies and provide a proof of principle for a potential new therapeutic strategy for treating B cell malignancies.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Macrófagos/citologia , Animais , Antineoplásicos Hormonais/uso terapêutico , Antineoplásicos Hormonais/toxicidade , Linhagem Celular Tumoral , Linhagem da Célula , Transdiferenciação Celular/efeitos dos fármacos , Humanos , Leucemia/metabolismo , Leucemia/patologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/metabolismo , Linfoma de Células B/mortalidade , Macrófagos/metabolismo , Camundongos , Fagocitose , Tamoxifeno/uso terapêutico , Tamoxifeno/toxicidade , Transcriptoma , Transplante HeterólogoRESUMO
Using high-resolution genomic microarray analysis, a distinct genomic profile was defined in 114 samples from patients with splenic marginal zone lymphoma (SMZL). Deletion or uniparental disomy of chromosome 7q were detected in 42 of 114 (37%) SMZLs but in only nine of 170 (5%) mature B-cell lymphomas (P < 0·00001). The presence of unmutated IGHV, genomic complexity, 17p13-TP53 deletion and 8q-MYC gain, but not 7q deletion, correlated with shorter overall survival of SMZL patients. Mapping studies narrowed down a commonly deleted region of 2·7 Mb in 7q32.1-q32.2 spanning a region between the SND1 and COPG2 genes. High-throughput sequencing analysis of the 7q32-deleted segment did not identify biallelic deletions/insertions or clear pathogenic gene mutations, but detected six nucleotide changes in IRF5 (n = 2), TMEM209 (n = 2), CALU (n = 1) and ZC3HC1 (n = 1) not found in healthy individuals. Comparative expression analysis found a fourfold down-regulation of IRF5 gene in lymphomas with 7q32 deletion versus non-deleted tumours (P = 0·032). Ectopic expression of IRF5 in marginal-zone lymphoma cells decreased proliferation and increased apoptosis in vitro, and impaired lymphoma development in vivo. These results show that cryptic deletions, insertions and/or point mutations inactivating genes within 7q32 are not common in SMZL, and suggest that IRF5 may be a haploinsufficient tumour suppressor in this lymphoma entity.
Assuntos
Cromossomos Humanos Par 7/genética , Genes Supressores de Tumor , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Fatores Reguladores de Interferon/genética , Linfoma de Zona Marginal Tipo Células B/genética , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Deleção de Sequência , Neoplasias Esplênicas/genética , Animais , Apoptose/genética , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/transplante , Cromossomos Humanos Par 7/ultraestrutura , Hibridização Genômica Comparativa , Regulação Neoplásica da Expressão Gênica , Genes de Imunoglobulinas , Humanos , Fatores Reguladores de Interferon/biossíntese , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/fisiologia , Estimativa de Kaplan-Meier , Linfoma de Zona Marginal Tipo Células B/mortalidade , Linfoma de Zona Marginal Tipo Células B/patologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/fisiologia , Mutação Puntual , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Esplênicas/mortalidade , Neoplasias Esplênicas/patologia , Translocação GenéticaRESUMO
BACKGROUND: LMO2 is highly expressed at the most immature stages of lymphopoiesis. In T-lymphocytes, aberrant LMO2 expression beyond those stages leads to T-cell acute lymphoblastic leukemia, while in B cells LMO2 is also expressed in germinal center lymphocytes and diffuse large B-cell lymphomas, where it predicts better clinical outcome. The implication of LMO2 in B-cell acute lymphoblastic leukemia must still be explored. DESIGN AND METHODS: We measured LMO2 expression by real time RT-PCR in 247 acute lymphoblastic leukemia patient samples with cytogenetic data (144 of them also with survival and immunophenotypical data) and in normal hematopoietic and lymphoid cells. RESULTS: B-cell acute lymphoblastic leukemia cases expressed variable levels of LMO2 depending on immunophenotypical and cytogenetic features. Thus, the most immature subtype, pro-B cells, displayed three-fold higher LMO2 expression than pre-B cells, common-CD10+ or mature subtypes. Additionally, cases with TEL-AML1 or MLL rearrangements exhibited two-fold higher LMO2 expression compared to cases with BCR-ABL rearrangements or hyperdyploid karyotype. Clinically, high LMO2 expression correlated with better overall survival in adult patients (5-year survival rate 64.8% (42.5%-87.1%) vs. 25.8% (10.9%-40.7%), P= 0.001) and constituted a favorable independent prognostic factor in B-ALL with normal karyotype: 5-year survival rate 80.3% (66.4%-94.2%) vs. 63.0% (46.1%-79.9%) (P= 0.043). CONCLUSIONS: Our data indicate that LMO2 expression depends on the molecular features and the differentiation stage of B-cell acute lymphoblastic leukemia cells. Furthermore, assessment of LMO2 expression in adult patients with a normal karyotype, a group which lacks molecular prognostic factors, could be of clinical relevance.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Metaloproteínas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Criança , Pré-Escolar , Humanos , Imunofenotipagem , Lactente , Cariotipagem , Proteínas com Domínio LIM , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Prognóstico , Proteínas Proto-Oncogênicas , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
In Burkitt lymphoma/leukemia (BL), achievement of complete remission with first-line chemotherapy remains a challenging issue, as most patients who respond remain disease-free, whereas those refractory have few options of being rescued with salvage therapies. The mechanisms underlying BL chemoresistance and how it can be circumvented remain undetermined. We previously reported the frequent inactivation of the proapoptotic BIM gene in B-cell lymphomas. Here we show that BIM epigenetic silencing by concurrent promoter hypermethylation and deacetylation occurs frequently in primary BL samples and BL-derived cell lines. Remarkably, patients with BL with hypermethylated BIM presented lower complete remission rate (24% vs 79%; P = .002) and shorter overall survival (P = .007) than those with BIM-expressing lymphomas, indicating that BIM transcriptional repression may mediate tumor chemoresistance. Accordingly, by combining in vitro and in vivo studies of human BL-xenografts grown in immunodeficient RAG2(-/-)γc(-/-) mice and of murine B220(+)IgM(+) B-cell lymphomas generated in Eµ-MYC and Eµ-MYC-BIM(+/-) transgenes, we demonstrate that lymphoma chemoresistance is dictated by BIM gene dosage and is reversible on BIM reactivation by genetic manipulation or after treatment with histone-deacetylase inhibitors. We suggest that the combination of histone-deacetylase inhibitors and high-dose chemotherapy may overcome chemoresistance, achieve durable remission, and improve survival of patients with BL.