RESUMEN
TCF3 is a lymphopoietic transcription factor that acquires somatic driver mutations in diffuse large B-cell lymphoma (DLBCL). Hypothesizing that expression patterns of TCF3-regulated genes can inform clinical management, we found that unsupervised clustering analysis with 15 TCF3-regulated genes and eight additional ones resolved local DLBCL cases into two main clusters, denoted Groups A and B, of which Group A manifested inferior overall survival (OS, p = 0.0005). We trained a machine learning model to classify samples into the Groups based on expression of the 23 transcripts in an independent validation cohort of 569 R-CHOP-treated DLBCL cases. Group A overlapped with the ABC cell-of-origin subgroup but its prognostic power was superior. GSEA analysis demonstrated asymmetric expression of 30 gene sets between the Groups, pointing to biological differences. We present, validate and make available a novel method to assign DLBCL cases into biologically-distinct groups with divergent OS following R-CHOP therapy.
Asunto(s)
Linfoma de Células B Grandes Difuso , Humanos , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ciclofosfamida/uso terapéutico , Doxorrubicina/uso terapéutico , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Prednisona/uso terapéutico , Pronóstico , Rituximab/uso terapéutico , Regulación hacia Arriba , Vincristina/uso terapéuticoRESUMEN
The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved ΦXXΦΦ motifs (with Φ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2A-PBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2A-AD1(1-37) complex indicated that E2A-AD1 adopts an α-helical structure and uses its ΦXXΦΦ motif to bind TAZ2. Whereas this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2.
Asunto(s)
Proteína de Unión a CREB/química , Proteína p300 Asociada a E1A/genética , Dominios Proteicos/genética , Factor de Transcripción 3/química , Linfocitos B/química , Linfocitos B/metabolismo , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/ultraestructura , Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/ultraestructura , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/ultraestructura , Humanos , Mutación/genética , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/ultraestructura , Unión Proteica/genética , Conformación Proteica , Factor de Transcripción 3/genética , Factor de Transcripción 3/ultraestructuraRESUMEN
Up-regulation of BCL2 in cases of diffuse large B-cell lymphoma (DLBCL) can confer treatment resistance. Quantitative immunofluorescence (QIF) histology allows objective quantification of protein-based biomarkers. We investigated the utility of QIF for evaluating BCL2 as a biomarker in DLBCL by quantifying BCL2 selectively in CD20-expressing lymphoma cells in biopsy samples from 116 cases of DLBCL in two cohorts one of which consisted of relapsed/refractory cases from a clinical trial. BCL2 protein by QIF correlated with BCL2 mRNA abundance and was associated with both translocation and copy number gain of the BCL2 gene. Elevated BCL2 protein expression by QIF, but not immunohistochemistry or mRNA quantification, was associated with inferior overall and relapse-free survival in the relapsed/refractory cohort. QIF is an effective means of quantifying BCL2 protein objectively in routine cancer biopsy specimens and shows promise for identifying relapsed/refractory DLBCL patients at risk of inferior outcomes after salvage therapy.
Asunto(s)
Linfoma de Células B Grandes Difuso , Proteínas Proto-Oncogénicas c-myc , Biopsia , Técnica del Anticuerpo Fluorescente , Humanos , Linfoma de Células B Grandes Difuso/genética , Recurrencia Local de Neoplasia , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.
Asunto(s)
Biomarcadores/análisis , Técnica del Anticuerpo Fluorescente , Immunoblotting/métodos , Proteínas/análisis , Línea Celular , Humanos , Estándares de Referencia , Reproducibilidad de los Resultados , Factores de Tiempo , Fijación del TejidoRESUMEN
BACKGROUND: We report the first case of composite lymphoma consisting of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma (FL) and high-grade B-cell lymphoma with MYC and BCL2 rearrangements within the same needle biopsy in which a clonal relationship between the FL and high-grade B-cell lymphoma components was demonstrated by molecular cytogenetics. CASE PRESENTATION: An 85-year-old man presented with masses in his neck and right groin. Cutting needle biopsy of the inguinal mass revealed the three lymphoma types which were morphologically, immunophenotypically and topographically distinct. Fluorescence in situ hybridization (FISH) identified an IGH-BCL2 rearrangement in both the FL and high-grade B-cell components while a MYC rearrangement was detected in the high-grade B-cell component alone. CONCLUSIONS: Our findings suggest that the high-grade lymphoma with MYC and BCL2 translocations evolved through transformation of the FL by a process that entailed acquisition of the MYC translocation. No clonal relationship between the FL and CLL/SLL components was evident since the IGH-BCL2 rearrangement was present in in the former but not the latter. This unique case of co-localized FL, CLL/SLL, and high-grade B-cell lymphoma contributes to our understanding of the clonal relationships that may exist between the components of composite lymphomas.
Asunto(s)
Linfoma Compuesto/genética , Linfoma Compuesto/patología , Linfoma Folicular/genética , Linfoma de Células B Grandes Difuso/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-myc/genética , Anciano de 80 o más Años , Reordenamiento Génico , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Linfoma Folicular/patología , Linfoma de Células B Grandes Difuso/patología , MasculinoRESUMEN
Although generally indolent, follicular lymphoma (FL) sometimes pursues a more aggressive course leading to early death. B-cell-specific Mo-MLV insertion site-1 (BMI1) is a member of the polycomb group (PcG) proteins that confer stem cell properties through gene silencing. We used multi-channel immunofluorescence and automated image analysis to quantify BMI1 selectively in the nuclei of FL-derived B-cells in routine biopsy specimens. Applying this assay to 109 pretreatment FL biopsy samples demonstrates a significant association between abundant BMI1 and reduced overall survival (p = .001); the statistically significant association with mortality persists in a Cox proportional hazards model that includes Follicular Lymphoma International Prognostic Index (FLIPI) score, histological grade, and the presence of a component of diffuse large B-cell lymphoma in the biopsy sample. Ascertaining BMI1 over-expression may be useful in identifying patients who might benefit from novel therapies directed at reversing the chromatin-modifying functions of BMI1.
Asunto(s)
Linfoma de Células B/metabolismo , Linfoma Folicular/tratamiento farmacológico , Complejo Represivo Polycomb 1/metabolismo , Rituximab/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Línea Celular Tumoral , Femenino , Células HeLa , Humanos , Células Jurkat , Células K562 , Estimación de Kaplan-Meier , Linfoma Folicular/metabolismo , Linfoma Folicular/patología , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud/métodos , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Pronóstico , Modelos de Riesgos ProporcionalesRESUMEN
The oncogenic transcription factor E2A-PBX1 is expressed consequent to chromosomal translocation 1;19 and is an important oncogenic driver in cases of pre-B-cell acute lymphoblastic leukemia (ALL). Elucidating the mechanism by which E2A-PBX1 induces lymphoid leukemia would be expedited by the availability of a tractable experimental model in which enforced expression of E2A-PBX1 in hematopoietic progenitors induces pre-B-cell ALL. However, hematopoietic reconstitution of irradiated mice with bone marrow infected with E2A-PBX1-expressing retroviruses consistently gives rise to myeloid, not lymphoid, leukemia. Here, we elucidate the hematopoietic consequences of forced E2A-PBX1 expression in primary murine hematopoietic progenitors. We show that introducing E2A-PBX1 into multipotent progenitors permits the retention of myeloid potential but imposes a dense barrier to lymphoid development prior to the common lymphoid progenitor stage, thus helping to explain the eventual development of myeloid, and not lymphoid, leukemia in transplanted mice. Our findings also indicate that E2A-PBX1 enforces the aberrant, persistent expression of some genes that would normally have been down-regulated in the subsequent course of hematopoietic maturation. We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself. Existing evidence suggests that the 1;19 translocation event takes place in committed B-lymphoid progenitors. However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis. Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.
Asunto(s)
Células Madre Hematopoyéticas/citología , Proteínas de Homeodominio/metabolismo , Linfopoyesis , Mielopoyesis , Proteínas de Fusión Oncogénica/metabolismo , Animales , Apoptosis , Células HEK293 , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Tejido Linfoide/citología , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas de Fusión Oncogénica/genética , Proto-Oncogenes Mas , Retroviridae/genéticaRESUMEN
OBJECTIVE: Interleukin-4 (IL-4) can induce macrophages to undergo alternative activation and polarize toward an M2-like or wound healing phenotype. Tumor associated macrophages (TAMs) are thought to assume M2-like properties, and it has been suggested they promote tumor growth and metastasis through effects on the tumor stroma, including extracelluar matrix remodeling and angiogenesis. IL-4 also promotes macrophage survival and formation of multinucleated giant cells, which have enhanced phagocytic behavior. This study was designed to explore the effect of cancer cell derived IL-4 on the tumor immune stroma and metastasis. METHODS: The metastatic mouse mammary carcinoma cell line AC2M2 was transduced with control or IL-4 encoding retroviruses and employed in orthotopic engraftment models. Tumor growth and metastasis were assessed. The cellular composition and biomarker expression of tumors were examined by immunohistochemical staining and flow cytometry; the transcriptome of the immune stroma was analyzed by nanoString based transcript quantitation; and in vivo and in vitro interactions between cancer cells and macrophages were assessed by flow cytometry and co-culture with video-time lapse microscopy, respectively. RESULTS: Unexpectedly, tumors from IL-4 expressing AC2M2 engrafted cells grew at reduced rates, and most surprising, they lost all metastatic potential relative to tumors from control AC2M2 cells. Myeloid cell numbers were not increased in IL-4 expressing tumors, but their expression of the M2 marker arginase I was elevated. Transcriptome analysis revealed an immune signature consistent with IL-4 induced M2 polarization of the tumor microenvironment and a generalized increase in myeloid involvement in the tumor stroma. Flow cytometry analysis indicated enhanced cancer cell phagocytosis by TAMs from IL-4 expressing tumors, and co-culture studies showed that IL-4 expressing cancer cells supported the survival and promoted the in vitro phagocytic behavior of macrophages. CONCLUSIONS: Although M2-like TAMs have been linked to enhanced tumorigenesis, this study shows that IL-4 production by cancer cells is associated with suppressed tumor growth and loss of metastatic potential as well as enhanced phagocytic behavior of TAMs.
RESUMEN
Follicular lymphoma (FL) is the most common indolent lymphoma. The vast majority of cases are associated with the chromosome translocation t(14;18), a somatic rearrangement that leads to constitutive expression of the anti-apoptotic BCL2 protein. Although t(14;18) clearly represents an important early event in FL pathogenesis, abundant evidence indicates that it is not sufficient. In particular, the recent application of next-generation DNA sequencing technology has uncovered numerous recurrent somatic genomic alterations associated with FL, most of which affect tumor suppressor genes (TSGs). In this article we review the existing literature on TSGs involved in the development and progression of FL. We consider the genes that are most frequently targeted by deleterious mutation, deletion or epigenetic silencing, along with strategies for developing new treatments that exploit the susceptibilities that may be conferred on lymphoma cells by the loss of particular TSGs.
Asunto(s)
Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Linfoma Folicular/genética , Linfoma Folicular/patología , Mutación/genética , Proteínas Supresoras de Tumor/genética , Humanos , PronósticoRESUMEN
We describe the first reported occurrence of a composite cutaneous lymphoma involving a mantle cell lymphoma (MCL) and primary cutaneous anaplastic large cell lymphoma. The lesion occurred in a 76-year-old man with longstanding MCL who developed nodular skin lesions on his trunk and extremities. Biopsy revealed a CD30-positive lymphoma with pathological features characteristic of cutaneous anaplastic large cell lymphoma in the superficial dermis and a subjacent deposit of MCL in the deep dermis and subcutaneous adipose tissue. Immunophenotyping demonstrated T versus B lymphoid origin, respectively, for the 2 neoplasms, and fluorescence in situ hybridization demonstrated an 11;14 chromosomal translocation exclusively in the MCL. These results argue that the lymphomas represented clonally distinct neoplasms. Our case illustrates the extreme diversity associated with the cutaneous manifestations of lymphoid neoplasia and in particular of composite lymphomas, which present diagnostic challenges for clinicians and pathologists alike.
Asunto(s)
Linfoma de Células del Manto/patología , Linfoma Anaplásico Cutáneo Primario de Células Grandes/patología , Neoplasias Complejas y Mixtas/patología , Neoplasias Cutáneas/patología , Anciano , Biomarcadores de Tumor/análisis , Humanos , Inmunohistoquímica , MasculinoRESUMEN
The E-protein transcription factors play essential roles in lymphopoiesis, with E12 and E47 (hereafter called E2A) being particularly important in B cell specification and maturation. The E2A gene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1. The two activation domains of E2A, AD1 and AD2, display redundant, independent, and cooperative functions in a cell-dependent manner. AD1 of E2A functions by binding the transcriptional co-activator CBP/p300; this interaction is required in oncogenesis and occurs between the conserved Ï-x-x-Ï-Ï motif in AD1 and the KIX domain of CBP/p300. However, co-activator recruitment by AD2 has not been characterized. Here, we demonstrate that the first of two conserved Ï-x-x-Ï-Ï motifs within AD2 of E2A interacts at the same binding site on KIX as AD1. Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation. Although AD2 is dispensable for oncogenesis, experimentally increasing the affinity of AD2 for KIX uncovered a latent potential to mediate immortalization of primary hematopoietic progenitors by E2A-PBX1. Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.
Asunto(s)
Factor de Transcripción 3/química , Activación Transcripcional , Factores de Transcripción p300-CBP/química , Sitios de Unión , Células de la Médula Ósea/metabolismo , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Factor de Transcripción 3/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
PURPOSE: Follicular lymphoma, the most common indolent lymphoma, is clinically heterogeneous. CDKN2A encodes the tumor suppressors p16(INK4a) and p14(ARF) and frequently suffers deleterious alterations in cancer. We investigated the hypothesis that deletion or hypermethylation of CDKN2A might identify follicular lymphoma cases with distinct clinical or pathologic features potentially amenable to tailored clinical management. EXPERIMENTAL DESIGN: Deletion of CDKN2A was detected in pretreatment biopsy specimens using a single nucleotide polymorphism-based approach or endpoint PCR, and methylation of CpG elements in CDKN2A was quantified by methylation-specific PCR. Correlations between CDKN2A status and pathologic or clinical characteristics, including overall survival (OS), were investigated in 106 cases using standard statistical methods. RESULTS: Deletion of CDKN2A was detected in 9 of 111 samples (8%) and methylation was detectable in 22 of 113 (19%). CDKN2A was either deleted or methylated in 29 of 106 cases (27%) and this status was associated with inferior OS especially among patients treated with rituximab (P = 0.004). CDKN2A deletion or methylation was associated with more advanced age (P = 0.012) and normal hemoglobin (P = 0.05) but not with sex, FLIPI score, ECOG stage, LDH, performance status, number of involved nodal sites, B symptoms, histologic grade, the presence of a component of diffuse large B-cell lymphoma, proliferation index, or other pathologic factors. CONCLUSIONS: Our results show that deletion or methylation of CDKN2A is relatively common in pretreatment follicular lymphoma biopsy specimens and defines a group of cases associated with reduced survival in the rituximab era presumably on the basis of more aggressive disease biology.
Asunto(s)
Metilación de ADN , Genes p16 , Linfoma Folicular/genética , Eliminación de Secuencia , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Silenciador del Gen , Humanos , Estimación de Kaplan-Meier , Captura por Microdisección con Láser , Linfoma Folicular/mortalidad , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Pronóstico , Modelos de Riesgos ProporcionalesRESUMEN
PURPOSE: Mantle-cell lymphoma (MCL) has a variable natural history but is incurable with current therapies. MicroRNAs (miRs) are useful in prognostic assessment of cancer. We determined an miR signature defining aggressiveness in B-cell non-Hodgkin lymphomas (NHL) and assessed whether this signature aids in MCL prognosis. METHODS: We assessed miR expression in a training set of 43 NHL cases. The miR signature was validated in 44 additional cases and examined on a training set of 119 MCL cases from four institutions in Canada. miRs significantly associated with overall survival were examined in an independent cohort of 114 MCL cases to determine association with patient outcome. miR expression was combined with current clinical prognostic factors to develop an enhanced prognostic model in patients with MCL. RESULTS: Fourteen miRs were differentially expressed between aggressive and indolent NHL; 11 of 14 were validated in an independent set of NHL (excluding MCL). miR-127-3p and miR-615-3p were significantly associated with overall survival in the MCL training set. Their expression was validated in an independent MCL patient set. In comparison with Ki-67, expression of these miRs was more significantly associated with overall survival among patients with MCL. miR-127-3p was combined with Ki-67 to create a new prognostic model for MCL. A similar model was created with miR-615-3p and Mantle Cell Lymphoma International Prognostic Index scores. CONCLUSION: Eleven miRs are differentially expressed between aggressive and indolent NHL. Two novel miRs were associated with overall survival in MCL and were combined with clinical prognostic models to generate novel prognostic data for patients with MCL.
Asunto(s)
Linfoma de Células B/genética , Linfoma de Células B/patología , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , MicroARNs/biosíntesis , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Femenino , Expresión Génica , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células del Manto/metabolismo , Masculino , MicroARNs/genética , Persona de Mediana Edad , Adhesión en Parafina , Pronóstico , Análisis de SupervivenciaRESUMEN
Recombinant interleukin-21 (IL-21) has potential utility in cancer therapy. Stimulation with IL-21 can induce apoptosis in follicular lymphoma (FL) cells, and existing studies have suggested that IL-21 signaling may function in tumor suppression. In order to elucidate the relationship between IL-21 receptor (IL-21R) expression and clinical and pathological features in FL, IL-21R was quantified in 114 pretreatment biopsy samples using either conventional immunohistochemistry or immunofluorescence microscopy and automated quantitative analysis (AQUA). Reduced expression of IL-21R was associated with favorable overall survival (p = 0.048). AQUA analysis showed an association with the presence of diffuse large B-cell lymphoma (DLBCL) in the biopsy sample (p = 0.03), and expression of IL-21R was up-regulated upon transformation of FL to DLBCL in two cases. Our results based on the largest survey to date raise the possibility that IL-21 signaling in FL cells, rather than being tumor suppressive, supports tumor progression and that therapeutic benefit could be realized by blocking IL-21R instead of stimulating it.
Asunto(s)
Linfoma Folicular/metabolismo , Linfoma Folicular/patología , Receptores de Interleucina-21/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Progresión de la Enfermedad , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , Linfoma Folicular/genética , Linfoma Folicular/mortalidad , Linfoma Folicular/terapia , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Receptores de Interleucina-21/genéticaRESUMEN
E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein-encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein-driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1-induced leukemia.
Asunto(s)
Proteínas de Homeodominio/química , Leucemia/genética , Proteínas de Fusión Oncogénica/química , Factores de Transcripción p300-CBP/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Transformación Celular Neoplásica/genética , Secuencia Conservada , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutación , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Unión Proteica/genética , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The E2A gene encodes the E-protein transcription factors E12 and E47 that play critical roles in B-lymphopoiesis. A somatic chromosomal translocation detectable in 5% of cases of acute lymphoblastic leukemia (ALL) involves E2A and results in expression of the oncogenic transcription factor E2A-PBX1. CREB binding protein (CBP) and its close paralog p300 are transcriptional co-activators with intrinsic histone acetyltransferase (HAT) activity. We and others have shown that direct binding of an N-terminal transcriptional activation domain present in E12/E47 and E2A-PBX1 to the KIX domain of CBP/p300 contributes to E2A protein function. In the current work we show for the first time that the catalytic HAT activity of CBP/p300 is increased in the presence of residues 1-483 of E2A (i.e., the portion present in E2A-PBX1). The addition of purified, recombinant E2A protein to in vitro assays results in a two-fold augmentation of CBP/p300 HAT activity, whereas in vivo assays show a ten-fold augmentation of HAT-dependent transcriptional induction and a five-fold augmentation of acetylation of reporter plasmid-associated histone by CBP in response to co-transfected E2A. Our results indicate that the HAT-enhancing effect is independent of the well-documented E2A-CBP interaction involving the KIX domain and suggest a role for direct, perhaps low affinity binding of E2A to a portion of CBP that includes the HAT domain and flanking elements. Our findings add to a growing body of literature indicating that interactions between CBP/p300 and transcription factors can function in a specific manner to modulate HAT catalytic activity.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteína de Unión a CREB , Histona Acetiltransferasas/metabolismo , Factores de Transcripción p300-CBP , Acetilación , Secuencia de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Células HEK293 , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas de Fusión Oncogénica/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Activación Transcripcional , Factores de Transcripción p300-CBP/metabolismoRESUMEN
E-proteins are basic helix-loop-helix transcription factors that function in cell type specification. The gene E2A encodes two E-proteins, E12 and E47, which are required in B-lymphopoiesis. E2A proteins can interact directly with the transcriptional co-activators and lysine acetyltranferases (KATs) CBP, p300 and PCAF to induce target gene transcription. Prior investigations have shown that the E2A-encoded isoform E2-5 is acetylated by CBP, p300 or PCAF in vitro or in vivo. However, E2-5 lacks the important N-terminal activation domain AD1. Furthermore, the acetylated residues in E-proteins have not been mapped, and the functional consequences of acetylation are largely unknown. Here, we use mutagenesis to show that a lysine residue at position 34 within AD1 of E12/E47 is acetylated by CBP/p300 and PCAF. Lys34 lies adjacent to a conserved helical LXXLL motif that interacts directly with the KIX domain of CBP/p300. We show that acetylation at Lys34 increases the affinity of AD1 for the KIX domain and enhances AD1-driven transcriptional induction. Our results illustrate for the first time that AD1 can both recruit, and be acetylated by, KATs and that KAT recruitment may promote transcriptional induction in part through acetylation of AD1 itself.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteína de Unión a CREB , Lisina , Factores de Transcripción p300-CBP , Acetilación , Secuencia de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/genética , Lisina/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Activación Transcripcional , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Follicular lymphoma, a relatively common neoplasm of mature B lymphocytes, generally pursues an indolent clinical course. The disease is biologically heterogeneous, however, and aggressive instances associated with short survival are relatively common. Because defects in the regulation of apoptotic cell death are fundamental in follicular lymphoma pathogenesis, we hypothesized that deregulated expression of components of the Rb signaling pathway may promote cell proliferation, thereby complementing antecedent antiapoptotic mutations and producing more aggressive disease. We determined the differential expression of key cell-cycle regulatory proteins in lymphoma cells by incorporating formalin-fixed, paraffin-embedded samples from the initial, diagnostic biopsies from 127 cases of follicular lymphoma into tissue microarrays, histologic sections of which were stained by immunohistochemistry for p53, pRb, p16(INK4a), and cyclin D3. The results were ascertained by visual inspection and then correlated with histopathological and clinical parameters, including overall survival. Our findings show that increased abundance of p53 or p16(INK4a) is associated with reduced overall survival and conventional pathological markers of tumor aggressiveness including high histologic grade. Therefore, subjective quantification of cell-cycle regulatory proteins by immunohistochemistry can identify biologically and clinically distinct subsets of follicular lymphoma cases.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Linfoma Folicular/clasificación , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Ciclo Celular/genética , Proliferación Celular , Humanos , Inmunohistoquímica , Linfoma Folicular/diagnóstico , Linfoma Folicular/metabolismo , Proteína de Retinoblastoma/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Childhood cancer is the leading cause of disease-related death in children aged 1-14 years in Canada and the USA and it has been hypothesized that transplacental exposure to environmental carcinogens such as benzene may contribute to the etiology of these cancers. Our objectives were to determine if transplacental benzene exposure increased tumor incidence in mouse offspring and assess fetal benzene metabolism capability. Pregnant CD-1 and C57Bl/6N mice were given intraperitoneal injections of corn oil, 200 mg/kg, or 400 mg/kg benzene on gestational days 8, 10, 12 and 14. A significant increase in tumor incidence was observed in CD-1, but not C57BL/6N, 1-year-old offspring exposed transplacentally to 200 mg/kg benzene. Hepatic and hematopoietic tumors were predominantly observed in male and female CD-1 offspring, respectively. Female CD-1 offspring exposed transplacentally to 200 mg/kg benzene had significantly suppressed bone marrow CD11b(+) cells 1 year after birth, correlating with reduced colony-forming unit granulocyte/macrophage numbers in 2-day-old pups. CD-1 and C57Bl/6N maternal blood benzene levels and fetal liver benzene, t, t-muconic acid, hydroquinone and catechol levels were analyzed by gas chromatography/mass spectrometry. Significant strain-, gender- and dose-related differences were observed. Male CD-1 fetuses had high hydroquinone levels, whereas females had high catechol levels after maternal exposure to 200 mg/kg benzene. This is the first demonstration that transplacental benzene exposure can induce hepatic and hematopoietic tumors in mice, which may be dependent on fetal benzene metabolism capability.