RESUMO
BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent and durable effects in B-cell malignancies. However, antigen loss or downregulation is a frequent cause of resistance. Here, we report development of a novel CAR T-cell therapy product to target CD79b, a pan B-cell antigen, widely expressed in most B-cell lymphomas. METHODS: We generated a novel anti-CD79b monoclonal antibody by hybridoma method. The specificity of the antibody was determined by testing against isogenic cell lines with human CD79b knock-in or knock-out. A single-chain variable fragment derived from the monoclonal antibody was used to make a panel of CD79b-targeting CAR molecules containing various hinge, transmembrane, and co-stimulatory domains. These were lentivirally transduced into primary T cells and tested for antitumor activity in in vitro and in vivo B-cell lymphoma models. RESULTS: We found that the novel anti-CD79b monoclonal antibody was highly specific and bound only to human CD79b and no other cell surface protein. In testing the various CD79b-targeting CAR molecules, superior antitumor efficacy in vitro and in vivo was found for a CAR consisting CD8α hinge and transmembrane domains, an OX40 co-stimulatory domain, and a CD3ζ signaling domain. This CD79b CAR specifically recognized human CD79b-expressing lymphoma cell lines but not CD79b knock-out cell lines. CD79b CAR T cells, generated from T cells from either healthy donors or patients with lymphoma, proliferated, produced cytokines, degranulated, and exhibited robust cytotoxic activity in vitro against CD19+ and CD19- lymphoma cell lines and patient-derived lymphoma tumors relapsing after prior CD19 CAR T-cell therapy. Furthermore, CD79b CAR T cells were highly efficient at eradicating pre-established lymphoma tumors in vivo in three aggressive lymphoma xenograft models, including two cell line-derived xenografts and one patient-derived xenograft. Notably, these CAR T cells did not demonstrate any significant tonic signaling activity or markers of exhaustion. CONCLUSION: Our results indicated that this novel CD79b CAR T-cell therapy product has robust antitumor activity against B-cell lymphomas. These results supported initiation of a phase 1 clinical trial to evaluate this product in patients with relapsed or refractory B-cell lymphomas.
Assuntos
Linfoma de Células B , Receptores de Antígenos Quiméricos , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Linfoma de Células B/tratamento farmacológico , Linfócitos T , Anticorpos Monoclonais/metabolismoAssuntos
Produtos Biológicos/administração & dosagem , Linfoma Difuso de Grandes Células B , Proteínas de Neoplasias/imunologia , Evasão Tumoral/efeitos dos fármacos , Adulto , Antígenos CD19/imunologia , Produtos Biológicos/efeitos adversos , Feminino , Humanos , Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Recidiva , Evasão Tumoral/genéticaRESUMO
Thoracic Aortic Aneurysms and Dissections (TAAD) are a major cause of death in the United States. The spectrum of TAAD ranges from genetic disorders, such as Marfan syndrome, to sporadic isolated disease of unknown cause. We hypothesized that genomic copy number variants (CNVs) contribute causally to early onset TAAD (ETAAD). We conducted a genome-wide SNP array analysis of ETAAD patients of European descent who were enrolled in the National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions (GenTAC). Genotyping was performed on the Illumina Omni-Express platform, using PennCNV, Nexus and CNVPartition for CNV detection. ETAAD patients (n = 108, 100% European American, 28% female, average age 20 years, 55% with bicuspid aortic valves) were compared to 7013 dbGAP controls without a history of vascular disease using downsampled Omni 2.5 data. For comparison, 805 sporadic TAAD patients with late onset aortic disease (STAAD cohort) and 192 affected probands from families with at least two affected relatives (FTAAD cohort) from our institution were screened for additional CNVs at these loci with SNP arrays. We identified 47 recurrent CNV regions in the ETAAD, FTAAD and STAAD groups that were absent or extremely rare in controls. Nine rare CNVs that were either very large (>1 Mb) or shared by ETAAD and STAAD or FTAAD patients were also identified. Four rare CNVs involved genes that cause arterial aneurysms when mutated. The largest and most prevalent of the recurrent CNVs were at Xq28 (two duplications and two deletions) and 17q25.1 (three duplications). The percentage of individuals harboring rare CNVs was significantly greater in the ETAAD cohort (32%) than in the FTAAD (23%) or STAAD (17%) cohorts. We identified multiple loci affected by rare CNVs in one-third of ETAAD patients, confirming the genetic heterogeneity of TAAD. Alterations of candidate genes at these loci may contribute to the pathogenesis of TAAD.
Assuntos
Aneurisma da Aorta Torácica/genética , Dissecção Aórtica/genética , Valva Aórtica/anormalidades , Variações do Número de Cópias de DNA/genética , Predisposição Genética para Doença/genética , Doenças das Valvas Cardíacas/genética , Adolescente , Adulto , Aorta/patologia , Doença da Válvula Aórtica Bicúspide , Feminino , Genômica , Genótipo , Humanos , Masculino , Síndrome de Marfan/genética , Adulto JovemRESUMO
The ascending thoracic aorta is designed to withstand biomechanical forces from pulsatile blood. Thoracic aortic aneurysms and acute aortic dissections (TAADs) occur as a result of genetically triggered defects in aortic structure and a dysfunctional response to these forces. Here, we describe mutations in the forkhead transcription factor FOXE3 that predispose mutation-bearing individuals to TAAD. We performed exome sequencing of a large family with multiple members with TAADs and identified a rare variant in FOXE3 with an altered amino acid in the DNA-binding domain (p.Asp153His) that segregated with disease in this family. Additional pathogenic FOXE3 variants were identified in unrelated TAAD families. In mice, Foxe3 deficiency reduced smooth muscle cell (SMC) density and impaired SMC differentiation in the ascending aorta. Foxe3 expression was induced in aortic SMCs after transverse aortic constriction, and Foxe3 deficiency increased SMC apoptosis and ascending aortic rupture with increased aortic pressure. These phenotypes were rescued by inhibiting p53 activity, either by administration of a p53 inhibitor (pifithrin-α), or by crossing Foxe3-/- mice with p53-/- mice. Our data demonstrate that FOXE3 mutations lead to a reduced number of aortic SMCs during development and increased SMC apoptosis in the ascending aorta in response to increased biomechanical forces, thus defining an additional molecular pathway that leads to familial thoracic aortic disease.
Assuntos
Aneurisma da Aorta Torácica/genética , Dissecção Aórtica/genética , Fatores de Transcrição Forkhead/genética , Adulto , Dissecção Aórtica/metabolismo , Dissecção Aórtica/patologia , Animais , Aorta/metabolismo , Aorta/patologia , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Apoptose , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , Mutação de Sentido Incorreto , Miócitos de Músculo Liso/fisiologia , Linhagem , Proteína Supressora de Tumor p53/genética , Remodelação Vascular , Peixe-ZebraRESUMO
OBJECTIVE: Although hypertension is the most common risk factor for thoracic aortic diseases, it is not understood how increased pressures on the ascending aorta lead to aortic aneurysms. We investigated the role of angiotensin II type 1 receptor activation in ascending aortic remodeling in response to increased biomechanical forces using a transverse aortic constriction (TAC) mouse model. APPROACH AND RESULTS: Two weeks after TAC, the increased biomechanical pressures led to ascending aortic dilatation and thickening of the medial and adventitial layers of the aorta. There was significant adventitial hyperplasia and inflammatory responses in TAC ascending aortas were accompanied by increased adventitial collagen, elevated inflammatory and proliferative markers, and increased cell density attributable to accumulation of myofibroblasts and macrophages. Treatment with losartan significantly blocked TAC-induced vascular inflammation and macrophage accumulation. However, losartan only partially prevented TAC-induced adventitial hyperplasia, collagen accumulation, and ascending aortic dilatation. Increased Tgfb2 expression and phosphorylated-Smad2 staining in the medial layer of TAC ascending aortas were effectively blocked with losartan. In contrast, the increased Tgfb1 expression and adventitial phospho-Smad2 staining were only partially attenuated by losartan. In addition, losartan significantly blocked extracellular signal-regulated kinase activation and reactive oxygen species production in the TAC ascending aorta. CONCLUSIONS: Inhibition of the angiotensin II type 1 receptor using losartan significantly attenuated the vascular remodeling associated with TAC but did not completely block the increased transforming growth factor-ß1 expression, adventitial Smad2 signaling, and collagen accumulation. These results help to delineate the aortic transforming growth factor-ß signaling that is dependent and independent of the angiotensin II type 1 receptor after TAC.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Anti-Hipertensivos/farmacologia , Aorta/efeitos dos fármacos , Aneurisma da Aorta Torácica/prevenção & controle , Hipertensão/tratamento farmacológico , Losartan/farmacologia , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Aorta/cirurgia , Aneurisma da Aorta Torácica/etiologia , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Aneurisma da Aorta Torácica/fisiopatologia , Pressão Arterial , Fenômenos Biomecânicos , Colágeno/metabolismo , Constrição , Dilatação Patológica , Modelos Animais de Doenças , Ecocardiografia Doppler , Hipertensão/complicações , Hipertensão/metabolismo , Hipertensão/patologia , Hipertensão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor Tipo 1 de Angiotensina/metabolismo , Proteína Smad2/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The Notch pathway can have both oncogenic and tumor suppressor roles, depending on cell context. For example, Notch signaling promotes T cell differentiation and is leukemogenic in T cells, whereas it inhibits early B cell differentiation and acts as a tumor suppressor in B cell leukemia where it induces growth arrest and apoptosis. The regulatory mechanisms that contribute to these opposing roles are not understood. Aberrant promoter DNA methylation and histone modifications are associated with silencing of tumor suppressor genes and have been implicated in leukemogenesis. Using methylated CpG island amplification (MCA)/DNA promoter microarray, we identified Notch3 and Hes5 as hypermethylated in human B cell acute lymphoblastic leukemia (ALL). We investigated the methylation status of other Notch pathway genes by bisulfite pyrosequencing. Notch3, JAG1, Hes2, Hes4 and Hes5 were frequently hypermethylated in B leukemia cell lines and primary B-ALL, in contrast to T-ALL cell lines and patient samples. Aberrant methylation of Notch3 and Hes5 in B-ALL was associated with gene silencing and was accompanied by decrease of H3K4 trimethylation and H3K9 acetylation and gain of H3K9 trimethylation and H3K27 trimethylation. 5-aza-2'-deoxycytidine treatment restored Hes5 expression and decreased promoter hypermethylation in most leukemia cell lines and primary B-ALL samples. Restoration of Hes5 expression by lentiviral transduction resulted in growth arrest and apoptosis in Hes5 negative B-ALL cells but not in Hes5 expressing T-ALL cells. These data suggest that epigenetic modifications are implicated in silencing of tumor suppressor of Notch/Hes pathway in B-ALL.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Epigênese Genética , Leucemia de Células B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores Notch/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/metabolismo , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Decitabina , Epigênese Genética/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Leucemia de Células B/sangue , Leucemia de Células B/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch3 , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Serrate-Jagged , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/patologia , Transcrição Gênica/efeitos dos fármacos , VorinostatRESUMO
RATIONALE: Mutations in myosin heavy chain (MYH11) cause autosomal dominant inheritance of thoracic aortic aneurysms and dissections. At the same time, rare, nonsynonymous variants in MYH11 that are predicted to disrupt protein function but do not cause inherited aortic disease are common in the general population and the vascular disease risk associated with these variants is unknown. OBJECTIVE: To determine the consequences of the recurrent MYH11 rare variant, R247C, through functional studies in vitro and analysis of a knock-in mouse model with this specific variant, including assessment of aortic contraction, response to vascular injury, and phenotype of primary aortic smooth muscle cells (SMCs). METHODS AND RESULTS: The steady state ATPase activity (actin-activated) and the rates of phosphate and ADP release were lower for the R247C mutant myosin than for the wild-type, as was the rate of actin filament sliding in an in vitro motility assay. Myh11(R247C/R247C) mice exhibited normal growth, reproduction, and aortic histology but decreased aortic contraction. In response to vascular injury, Myh11(R247C/R247C) mice showed significantly increased neointimal formation due to increased SMC proliferation when compared with the wild-type mice. Primary aortic SMCs explanted from the Myh11(R247C/R247C) mice were dedifferentiated compared with wild-type SMCs based on increased proliferation and reduced expression of SMC contractile proteins. The mutant SMCs also displayed altered focal adhesions and decreased Rho activation, associated with decreased nuclear localization of myocardin-related transcription factor-A. Exposure of the Myh11(R247C/R247C) SMCs to a Rho activator rescued the dedifferentiated phenotype of the SMCs. CONCLUSIONS: These results indicate that a rare variant in MYH11, R247C, alters myosin contractile function and SMC phenotype, leading to increased proliferation in vitro and in response to vascular injury.
Assuntos
Músculo Liso Vascular/metabolismo , Mutação , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Vasoconstrição , Trifosfato de Adenosina/metabolismo , Animais , Aorta/metabolismo , Sítios de Ligação , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Adesões Focais/metabolismo , Adesões Focais/patologia , Técnicas de Introdução de Genes , Genótipo , Cinética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Cadeias Pesadas de Miosina/genética , Fenótipo , Transativadores/metabolismo , Transfecção , Vasoconstrição/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Chromosomal deletions or reciprocal duplications of the 16p13.1 region have been implicated in a variety of neuropsychiatric disorders such as autism, schizophrenia, epilepsies, and attention-deficit hyperactivity disorder (ADHD). In this study, we investigated the association of recurrent genomic copy number variants (CNVs) with thoracic aortic aneurysms and dissections (TAAD). By using SNP arrays to screen and comparative genomic hybridization microarrays to validate, we identified 16p13.1 duplications in 8 out of 765 patients of European descent with adult-onset TAAD compared with 4 of 4,569 controls matched for ethnicity (P = 5.0 × 10â»5, OR = 12.2). The findings were replicated in an independent cohort of 467 patients of European descent with TAAD (P = 0.005, OR = 14.7). Patients with 16p13.1 duplications were more likely to harbor a second rare CNV (P = 0.012) and to present with aortic dissections (P = 0.010) than patients without duplications. Duplications of 16p13.1 were identified in 2 of 130 patients with familial TAAD, but the duplications did not segregate with TAAD in the families. MYH11, a gene known to predispose to TAAD, lies in the duplicated region of 16p13.1, and increased MYH11 expression was found in aortic tissues from TAAD patients with 16p13.1 duplications compared with control aortas. These data suggest chromosome 16p13.1 duplications confer a risk for TAAD in addition to the established risk for neuropsychiatric disorders. It also indicates that recurrent CNVs may predispose to disorders involving more than one organ system, an observation critical to the understanding of the role of recurrent CNVs in human disease and a finding that may be common to other recurrent CNVs involving multiple genes.
Assuntos
Aneurisma da Aorta Torácica/genética , Dissecção Aórtica/genética , Duplicação Cromossômica/genética , Cromossomos Humanos Par 16/genética , Adulto , Idoso , Dissecção Aórtica/patologia , Aorta/patologia , Aneurisma da Aorta Torácica/patologia , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Linhagem , Fenótipo , Fatores de RiscoRESUMO
We performed a genome-wide analysis of aberrant DNA methylation in chronic lymphocytic leukemia (CLL) using methylated CpG island amplification (MCA) coupled with a promoter microarray. We identified 280 potential targets of aberrant DNA methylation in CLL. These genes were located more frequently in chromosomes 19 (16%, p=0.001), 16 (11%, p=0.001), 17 (10%, p=0.02) and 11 (9%, p=0.02) and could be grouped in several functional networks. Methylation status was confirmed for 22 of these genes (SOX11, DLX1, FAM62C, SOX14, RSPO1, ADCY5, HAND2,SPOCK, MLL, ING1, PRIMA1, BCL11B, LTBP2, BNC1, NR2F2, SALL1, GALGT2, LHX1, DLX4, KLK10, TFAP2 and APP) in 78 CLL patients by pyrosequencing. As a proof of principle, we analyzed the expression of 2 genes, PRIMA1 and APP, in primary cells and of GALGT2, TFAP2C and PRIMA1 in leukemia cells. There was an inverse association between methylation and gene expression. This could be reversed by treatment with 5-aza-2'-deoxycytidine in cell lines. Treatment in a clinical trial with 5-azacitidine resulted in decreased methylation of LINE, DLX4 and SALL1 in the peripheral blood B-cells of patients with CLL. IgVH mutational status or ZAP-70 expression were not associated with specific methylation profiles. By multivariate analysis, methylation of LINE and APP was associated with shorter overall survival (p = 0.045 and 0.0035, respectively). This study demonstrates that aberrant DNA methylation is common and has potential prognostic and therapeutic value in CLL.
Assuntos
Metilação de DNA , Epigênese Genética/fisiologia , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Adulto , Idoso , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Metilação de DNA/fisiologia , Regulação para Baixo/genética , Feminino , Seguimentos , Inativação Gênica/fisiologia , Estudo de Associação Genômica Ampla , Células HL-60 , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Análise em Microsséries/métodos , Pessoa de Meia-Idade , Prognóstico , Transdução de Sinais/genética , Estudos de Validação como AssuntoRESUMO
Tuberous sclerosis complex (TSC) is a genetic disorder with pleiotropic manifestations caused by heterozygous mutations in either TSC1 or TSC2. One of the less investigated complications of TSC is the formation of aneurysms of the descending aorta, which are characterized on pathologic examination by smooth muscle cell (SMC) proliferation in the aortic media. SMCs were explanted from Tsc2(+/-) mice to investigate the pathogenesis of aortic aneurysms caused by TSC2 mutations. Tsc2(+/-) SMCs demonstrated increased phosphorylation of mammalian target of rapamycin (mTOR), S6 and p70S6K and increased proliferation rates compared with wild-type (WT) SMCs. Tsc2(+/-) SMCs also had reduced expression of SMC contractile proteins compared with WT SMCs. An inhibitor of mTOR signaling, rapamycin, decreased SMC proliferation and increased contractile protein expression in the Tsc2(+/-) SMCs to levels similar to WT SMCs. Exposure to alpha-elastin fragments also decreased proliferation of Tsc2(+/-) SMCs and increased levels of p27(kip1), but failed to increase expression of contractile proteins. In response to artery injury using a carotid artery ligation model, Tsc2(+/-) mice significantly increased neointima formation compared with the control mice, and the neointima formation was inhibited by treatment with rapamycin. These results demonstrate that Tsc2 haploinsufficiency in SMCs increases proliferation and decreases contractile protein expression and suggest that the increased proliferative potential of the mutant cells may be suppressed in vivo by interaction with elastin. These findings provide insights into the molecular pathogenesis of aortic disease in TSC patients and identify a potential therapeutic target for treatment of this complication of the disease.
Assuntos
Aorta Torácica/patologia , Doenças Torácicas/genética , Doenças Torácicas/terapia , Proteínas Supressoras de Tumor/deficiência , Animais , Aorta Torácica/diagnóstico por imagem , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/patologia , Proliferação de Células/efeitos dos fármacos , Criança , Proteínas Contráteis/metabolismo , Elastina/farmacologia , Humanos , Angiografia por Ressonância Magnética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos , Mutação/genética , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Peptídeos/farmacologia , Fenótipo , Proteínas , Radiografia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Doenças Torácicas/diagnóstico , Fatores de Transcrição/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismo , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Eph receptors and their ephrin ligands are involved in normal hematopoietic development and tumorigenesis. Using methylated CpG island amplification/DNA promoter microarray, we identified several EPH receptor and EPHRIN genes as potential hypermethylation targets in acute lymphoblastic leukemia (ALL). We subsequently studied the DNA methylation status of the Eph/ephrin family by bisulfite pyrosequencing. Hypermethylation of EPHA2, -A4, -A5, -A6, -A7, -A10, EPHB1, -B2, -B3, -B4, EFNA1, -A3, -A5, and EFNB1 and -B2 genes was detected in leukemia cell lines and primary ALL bone marrow samples. Expression analysis of EPHB4, EFNB2, and EFNA5 genes demonstrated that DNA methylation was associated with gene silencing. We cloned the promoter region of EPHB4 and demonstrated that promoter hypermethylation can result in EPHB4 transcriptional silencing. Restoration of EPHB4 expression by lentiviral transduction resulted in reduced proliferation and apoptotic cell death in Raji cells in which EPHB4 is methylated and silenced. Finally, we demonstrated that phosphorylated Akt is down-regulated in Raji cells transduced with EPHB4. These results suggest that epigenetic silencing by hypermethylation of EPH/EPHRIN family genes contributes to ALL pathogenesis and that EPHB4 can function as a tumor suppressor in ALL.
Assuntos
Metilação de DNA , Efrina-B2/genética , Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptor EphB4/genética , Apoptose/fisiologia , Divisão Celular/fisiologia , Efrina-B2/metabolismo , Efrina-B2/farmacologia , Efrinas/genética , Efrinas/metabolismo , Efrinas/farmacologia , Inativação Gênica , Genes Supressores de Tumor , Humanos , Células Jurkat , Família Multigênica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prevalência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor EphB4/metabolismo , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Análise de Sobrevida , Transdução GenéticaRESUMO
Histone deacetylase (HDAC) inhibitors have been shown to induce cell cycle arrest, terminal differentiation, and apoptosis in a broad spectrum of human tumors and animal xenograft models. JNJ-26481585 is a hydroxamic acid derivative, second-generation pan-HDAC inhibitor that has demonstrated high potency in preclinical studies. In the current study, we demonstrated that JNJ-26481585 has antileukemia and molecular activity in leukemia cell lines and primary human leukemia cells. We also observed a synergistic effect between treatment with decitabine and JNJ-26481585. In conclusion, JNJ-26481585 is a potent second-generation pan-HDAC inhibitor with activity in human leukemia, and it is currently in clinical development.
Assuntos
Antineoplásicos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Leucemia/tratamento farmacológico , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Decitabina , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Inibidores de Histona Desacetilases , Humanos , Células Tumorais CultivadasRESUMO
Marfan syndrome (MFS) is an autosomal dominant condition with pleiotropic manifestations involving the skeletal, ocular, and cardiovascular systems. The diagnosis is based primarily on clinical involvement of these and other systems, referred to as the Ghent criteria. We have identified three Hispanic families from Mexico with cardiovascular and ocular manifestations due to novel FBN1 mutations but with paucity of skeletal features. The largest family, hMFS001, had a frameshift mutation in exon 24 (3075delC) identified as the cause of aortic disease in the family. Assessment of eight affected adults revealed no major skeletal manifestation of MFS. Family hMFS002 had a missense mutation (R1530C) in exon 37. Four members fulfilled the criteria for ocular and cardiovascular phenotype but lacked skeletal manifestations. Family hMFS003 had two consecutive missense FBN1 mutations (C515W and R516G) in exon 12. Eight members fulfilled the ocular criteria for MFS and two members had major cardiovascular manifestations, however none of them met criteria for skeletal system. These data suggest that individuals of Hispanic descent with FBN1 mutations may not manifest skeletal features of the MFS to the same extent as Caucasians. We recommend that echocardiogram, ocular examination and FBN1 molecular testing be considered for any patients with possible MFS even in the absence of skeletal features, including Hispanic patients.
Assuntos
Síndrome de Marfan/genética , Proteínas dos Microfilamentos/genética , Mutação de Sentido Incorreto , Adulto , Doenças Cardiovasculares/genética , Análise Mutacional de DNA , Éxons , Saúde da Família , Feminino , Fibrilina-1 , Fibrilinas , Humanos , Masculino , Síndrome de Marfan/etnologia , México , Modelos Genéticos , Mutação , Linhagem , FenótipoRESUMO
Steroid receptor coactivator-1 (SRC-1) is a coactivator for nuclear hormone receptors such as estrogen and progesterone receptors and certain other transcription factors such as Ets-2 and PEA3. SRC-1 expression in breast cancer is associated with HER2 and c-Myc expression and with reduced disease-free survival. In this study, SRC-1(-/-) mice were backcrossed with FVB mice and then cross-bred with MMTV-polyoma middle T antigen (PyMT) mice to investigate the role of SRC-1 in breast cancer. Although mammary tumor initiation and growth were similar in SRC-1(-/-)/PyMT and wild-type (WT)/PyMT mice, genetic ablation of SRC-1 antagonized PyMT-induced restriction of mammary ductal differentiation and elongation. SRC-1(-/-)/PyMT mammary tumors were also more differentiated than WT/PyMT mammary tumors. The intravasation of mammary tumor cells and the frequency and extent of lung metastasis were drastically reduced in SRC-1(-/-)/PyMT mice compared with WT/PyMT mice. Metastatic analysis of transplanted WT/PyMT and SRC-1(-/-)/PyMT tumors in SRC-1(-/-) and WT recipient mice revealed that SRC-1 played an intrinsic role in tumor cell metastasis. Furthermore, SRC-1 was up-regulated during mammary tumor progression. Disruption of SRC-1 inhibited Ets-2-mediated HER2 expression and PyMT-stimulated Akt activation in the mammary tumors. Disruption of SRC-1 also suppressed colony-stimulating factor-1 (CSF-1) expression and reduced macrophage recruitment to the tumor site. These results suggest that SRC-1 specifically promotes metastasis without affecting primary tumor growth. SRC-1 may promote metastasis through mediating Ets-2-mediated HER2 expression and activating CSF-1 expression for macrophage recruitment. Therefore, functional interventions for coactivators like SRC-1 may provide unique approaches to control breast cancer progression and metastasis.
Assuntos
Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Histona Acetiltransferases/genética , Metástase Neoplásica/patologia , Fatores de Transcrição/genética , Animais , Neoplasias da Mama/etiologia , Neoplasias da Mama/genética , Diferenciação Celular , Feminino , Fator Estimulador de Colônias de Macrófagos/genética , Camundongos , Camundongos Knockout , Metástase Neoplásica/genética , Transplante de Neoplasias , Coativador 1 de Receptor Nuclear , Proteína Proto-Oncogênica c-ets-2/genética , Receptor ErbB-2/genéticaRESUMO
The term epigenetics refers to the study of a number of biochemical modifications of chromatin that have an impact on gene expression regulation. Aberrant epigenetic lesions, in particular DNA methylation of promoter associated CpG islands, are common in acute lymphocytic leukemia (ALL). Recent data from multiple laboratories indicate that several hundred genes, involving dozens of critical molecular pathways, are epigenetically suppressed in ALL. Because these lesions are potentially reversible, the reactivation of these pathways using, for instance, hypomethylating agents may have therapeutic potential in this disease. Furthermore, the analysis of epigenetic alterations in ALL may allow: (1) identification of subsets of patients with poor prognosis when treated with conventional therapy; (2) development of new techniques to evaluate minimal residual disease; (3) better understanding of the differences between pediatric and adult ALL; and (4) new therapeutic interventions by incorporating agents with hypomethylating activity to conventional chemotherapeutic programs. In this review, we describe the role of epigenetic alterations in ALL from a translational perspective.
Assuntos
Epigênese Genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adulto , Fatores Etários , Criança , Pré-Escolar , Metilação de DNA , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , PrognósticoRESUMO
Activating signal cointegrator 2 (ASC-2) is a transcriptional coactivator of many nuclear receptors (NRs) and other transcription factors and contains two NR-interacting LXXLL motifs (NR boxes). In the pancreas, ASC-2 is expressed only in the endocrine cells of the islets of Langerhans, but not in the exocrine cells. Thus, we examined the potential role of ASC-2 in insulin secretion from pancreatic beta-cells. Overexpressed ASC-2 increased glucose-elicited insulin secretion, whereas insulin secretion was decreased in islets from ASC-2+/- mice. DN1 and DN2 are two dominant-negative fragments of ASC-2 that contain NR boxes 1 and 2, respectively, and block the interactions of cognate NRs with the endogenous ASC-2. Primary rat islets ectopically expressing DN1 or DN2 exhibited decreased insulin secretion. Furthermore, relative to the wild type, ASC-2+/- mice showed reduced islet mass and number, which correlated with increased apoptosis and decreased proliferation of ASC-2+/- islets. These results suggest that ASC-2 regulates insulin secretion and beta-cell survival and that the regulatory role of ASC-2 in insulin secretion appears to involve, at least in part, its interaction with NRs via its two NR boxes.
Assuntos
Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Proliferação de Células , DNA/genética , Expressão Gênica , Glucose/metabolismo , Glucose/farmacologia , Técnicas In Vitro , Secreção de Insulina , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Coativadores de Receptor Nuclear , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos OLETF , Ratos Mutantes , Ratos Sprague-DawleyRESUMO
Amplified in breast cancer 1 (AIB1; steroid receptor coactivator-3, p/CIP, RAC3, ACTR, TRAM-1, or NCoA-3) is a transcriptional coactivator for nuclear receptors and certain other transcription factors and is a newly defined oncogene overexpressed in human breast cancer. Although the role and molecular mechanism of AIB1 in normal physiology and in breast cancer are currently under intensive investigation, the role of AIB1 in determination of the susceptibility of mammary gland to chemical carcinogens remains uncharacterized. In this study, we used back-crossed FVB wild-type (WT) and AIB1 mutant mice to assess the role of AIB1 in mammary gland development and in carcinogen-induced tumorigenesis. We show that mammary ductal growth was delayed in AIB1-/- mice with FVB strain background, and mammary ductal outgrowths emanating from the AIB1-/- mammary epithelial transplants in WT mice also were attenuated, indicating that the role of AIB1 in mammary ductal growth is a mammary epithelial autonomous function. In mice treated with the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), AIB1 deficiency protected the mammary gland, but not the skin, from tumorigenesis. AIB1 deficiency suppressed the up-regulation of the insulin receptor substrate (IRS)-1 and IRS-2 and thereby inhibited the activation of Akt, expression of cyclin D1, and cell proliferation. The suppression of these components for insulin-like growth factor-I signaling might be partially responsible for the decreased DMBA-induced mammary tumor initiation and progression in AIB1-/- mice. Our results suggest that AIB1 may serve as a potential target for prevention of carcinogen-induced breast cancer initiation and for treatment of breast cancer progression.
Assuntos
Neoplasias Mamárias Experimentais/prevenção & controle , Fatores de Transcrição/deficiência , 9,10-Dimetil-1,2-benzantraceno , Animais , Carcinógenos , Processos de Crescimento Celular/fisiologia , Ciclina D1/metabolismo , Feminino , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Coativador 3 de Receptor Nuclear , Hipófise/transplante , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The amplified-in-breast cancer 3 (AIB3) is a nuclear receptor coactivator amplified and overexpressed in human breast cancers. AIB3(-/-) mice die during gestation, whereas AIB3(+/-) mice exhibit normal development. Here, we demonstrate that AIB3 protein is mainly located in the nuclei of mammary epithelial cells and tumor cells and its levels are elevated in mammary epithelial cells at middle pregnant stage and in mammary tumor cells. To examine whether AIB3 reduction affects mammary tumorigenesis, we generated wild-type mouse mammary tumor virus/polyoma middle-T (WT/PyMT) and AIB3(+/-)/PyMT mice. Mammary tumor development in AIB3(+/-)/PyMT female and male mice was substantially accelerated compared with that in WT/PyMT mice, because of increased cell proliferation in early tumorigenic lesions, including ductal hyperplasia and mammary intraepithelial neoplasia. Tumor formation in nude mice that received premalignant AIB3(+/-)/PyMT mammary tissue was much faster than in nude mice that received transplants of premalignant WT/PyMT mammary tissue, which indicated that the accelerated tumorigenesis in AIB3(+/-)/PyMT mammary glands is due to a mammary epithelial autonomous defect. Expression of PyMT, estrogen receptor alpha and estrogen receptor alpha-regulated genes was unaffected in AIB3(+/-)/PyMT mammary glands, which suggests that the acceleration of mammary tumor formation in AIB3(+/-)/PyMT mice was not a consequence of changes in PyMT expression or in estrogen receptor function. Importantly, the inhibitory effects of peroxisome proliferator-activated receptor gamma (PPARgamma) and retinoid-X receptor (RXR) ligands on AIB3(+/-)/PyMT cell proliferation and the transcriptional function of PPARgamma in AIB3(+/-)/PyMT cells were reduced. Thus, AIB3 haplodeficiency may facilitate PyMT-induced tumorigenesis through a partial impairment of PPARgamma and RXR function. These results suggest that AIB3 may be a tumor suppressor that is required for the inhibition of cell proliferation by PPARgamma and RXR.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Neoplasias Mamárias Experimentais/patologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores do Ácido Retinoico/fisiologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/fisiologia , Animais , Divisão Celular , Transformação Celular Neoplásica/patologia , Células Epiteliais/metabolismo , Feminino , Haploidia , Imuno-Histoquímica , Ligantes , Masculino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Although the amplified in breast cancer 1 (AIB1) coactivator is amplified and overexpressed in breast cancers, its role in mammary carcinogenesis remains unknown. We demonstrate that during mammary development and tumorigenesis, the elevation of AIB1 level and its nuclear localization correlate with normal and transformed mammary epithelial proliferation, whereas its lower expression and cytoplasmic localization correlate with mammary epithelial quiescence and differentiation. In this study, the role of AIB1 in breast tumor initiation, progression, and metastasis was studied by generating AIB1(+/+), AIB1(+/-), and AIB1(-/-) mice harboring the mouse mammary tumor virus/v-Ha-ras (ras) transgene that induces breast tumors. Breast tumor incidence was reduced dramatically in the intact AIB1(-/-)-ras virgin mice and inhibited completely in the ovariectomized AIB1(-/-)-ras mice. Breast tumor latency was delayed significantly in AIB1(-/-)-ras virgin mice with natural estrous cycles, multiparous mice with cyclically elevated reproductive hormones, and virgin mice bearing pituitary isografts with persistently elevated hormones. Although AIB1 deficiency significantly suppressed mammary tumorigenesis under all of the concentrations of ovarian hormones, it did not affect the promotional role of ovarian hormones on mammary tumorigenesis, suggesting that AIB1 and ovarian hormones contribute to mammary carcinogenesis through different pathways. AIB1 deficiency did not alter the expression of estrogen and progesterone-responsive genes in the mammary gland, but it caused partial resistance to the insulin-like growth factor I because of a significant reduction in the insulin receptor substrates. The impaired insulin-like growth factor I pathway in AIB1(-/-)-ras mammary epithelium and tumor cells was responsible in part for the suppression of mammary tumorigenesis and metastasis caused by inhibition of cell proliferation and migration. These results suggest that a more effective strategy to control breast cancer is to target AIB1-mediated and ovarian hormone-initiated pathways.
Assuntos
Neoplasias da Mama/prevenção & controle , Genes ras , Fator de Crescimento Insulin-Like I/fisiologia , Transdução de Sinais , Fatores de Transcrição/fisiologia , Animais , Divisão Celular , Progressão da Doença , Ativação Enzimática , Receptor alfa de Estrogênio , Feminino , Histona Acetiltransferases , Neoplasias Pulmonares/secundário , Glândulas Mamárias Animais/química , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Coativador 3 de Receptor Nuclear , Ovariectomia , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Transativadores/fisiologia , Fatores de Transcrição/análise , Fatores de Transcrição/deficiênciaRESUMO
Transcriptional activities of nuclear receptors are modulated by coactivators and corepressors. The amplified in breast cancer-3 protein (AIB3, also known as ASC-2, RAP250, PRIP, TRBP, and NCR) is a newly identified nuclear receptor coactivator that is amplified and overexpressed in breast cancers. This study aims to investigate the spatial expression of AIB3 mRNA and protein in various murine tissues. Quantitative measurements revealed that the concentrations of AIB3 mRNA differ substantially in different tissues in a descending order from the following: testis, brain, thymus, white fat, pituitary, ovary, adrenal gland, lung, uterus, kidney, heart, skeletal muscle, liver, and virgin mammary gland. The AIB3 mRNA level in the testis is 165-fold higher than that in the virgin mammary gland. Specific antiserum was generated and used to map the distribution of AIB3 protein by immunohistochemistry. Although AIB3 protein was detected in many tissues, the AIB3 immunoreactivities varied significantly from cell type to cell type. High levels of AIB3 immunoreactivity were observed in hormone target cells including the testicular Sertoli cells, follicular granulosa cells, and epithelial cells of the prostate, uterus, mammary gland, and kidney tubules. Medium and low levels of AIB3 immunoreactivities were also detected in a variety of other cell types. These results demonstrate that AIB3 mRNA and protein are preferentially expressed in specific cell types, suggesting that AIB3 may support the function of nuclear receptors in a cell type-specific manner.