RESUMEN
Multiple myeloma (MM) remains incurable, and treatment of relapsed/refractory (R/R) disease is challenging. There is an unmet need for more targeted therapies in this setting; deep cellular and molecular phenotyping of the tumor and microenvironment in MM could help guide such therapies. This phase 1b study (NCT02431208) evaluated the safety and efficacy of the anti-programmed death-ligand 1 monoclonal antibody atezolizumab (Atezo) alone or in combination with the standard of care (SoC) treatments lenalidomide (Len) or pomalidomide (Pom) and/or daratumumab (Dara) in patients with R/R MM. Study endpoints included incidence of adverse events (AEs) and overall response rate (ORR). A novel unsupervised integrative multi-omic analysis was performed using RNA sequencing, mass cytometry immunophenotyping, and proteomic profiling of baseline and on-treatment bone marrow samples from patients receiving Atezo monotherapy or Atezo+Dara. A similarity network fusion (SNF) algorithm was applied to preprocessed data. Eighty-five patients were enrolled. Treatment-emergent deaths occurred in 2 patients; both deaths were considered unrelated to study treatment. ORRs ranged from 11.1% (Atezo+Len cohorts, n=18) to 83.3% (Atezo+Dara+Pom cohort, n=6). High-dimensional multi-omic profiling of the tumor microenvironment and integrative SNF analysis revealed novel correlations between cellular and molecular features of the tumor and immune microenvironment, patient selection criteria, and clinical outcome. Atezo monotherapy and SoC combinations were safe in this patient population and demonstrated some evidence of clinical efficacy. Integrative analysis of high dimensional genomics and immune data identified novel clinical correlations that may inform patient selection criteria and outcome assessment in future immunotherapy studies for myeloma.
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Mieloma Múltiple , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Microambiente Tumoral , Multiómica , Proteómica , Lenalidomida/uso terapéuticoRESUMEN
Rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) is the current standard therapy for patients with diffuse large B-cell lymphoma (DLBCL) and is curative in â¼60% of patients. Atezolizumab is a humanized immunoglobulin G1 monoclonal antibody that targets programmed death-ligand 1 and has previously shown antitumor activity in several tumor types. In a phase 1b/2 trial (NCT02596971), we evaluated the safety and efficacy of atezolizumab in combination with R-CHOP (atezo-R-CHOP; for 6-8 cycles) in patients with previously untreated DLBCL. Patients achieving a complete response (CR) at the end of induction received consolidation therapy with atezolizumab on day 1 of each 21-day cycle for an additional 17 cycles. Overall, 42 patients with DLBCL were included in this analysis. The primary endpoint, CR rate at the end of induction, as assessed by an independent review committee (modified Lugano 2014 criteria), was 77.5% (95% confidence interval [CI], 64.0-87.7; n = 40). Investigator-assessed progression-free survival and overall survival at 3 years were 77.4% (95% CI, 59.7-88.0) and 87.2% (95% CI, 71.9-94.5), respectively. All treated patients experienced ≥1 adverse event (AE; 32 patients [76.2%] had grade 3-4 AE). One patient had a fatal AE (unconfirmed progressive multifocal leukoencephalopathy) that was considered related to atezolizumab and rituximab, and 17 patients (40.5%) experienced atezolizumab-related AEs of special interest. In previously untreated patients with DLBCL, atezo-R-CHOP demonstrated encouraging clinical efficacy and a safety profile consistent with the known toxicities of the individual drugs. This trial was registered at www.clinicaltrials.gov as #NCT02596971.
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Linfoma de Células B Grandes Difuso , Humanos , Rituximab/efectos adversos , Anticuerpos Monoclonales de Origen Murino , Anticuerpos Monoclonales Humanizados/efectos adversos , Vincristina/efectos adversos , Ciclofosfamida/efectos adversos , Doxorrubicina/efectos adversos , Prednisona/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversosRESUMEN
Obinutuzumab (G) chemoimmunotherapy demonstrated improved progression-free survival (PFS) vs rituximab-based chemoimmunotherapy in patients with previously untreated follicular lymphoma (FL) in the GALLIUM trial. Atezolizumab (atezo) is a programmed death-ligand 1 inhibitor with a complementary mechanism of action to G by restoring cytotoxic T-cell function. We evaluated the safety and efficacy of atezo-G-bendamustine in patients with previously untreated FL in a phase Ib/II trial (#NCT02596971). A safety run-in phase was followed by an expansion phase with atezo-G-bendamustine induction and atezo-G maintenance for ≤24 months. Forty patients with previously untreated FL were enrolled and treated with atezo-G-bendamustine. The primary endpoint, complete response (CR) rate, assessed by an independent review committee (IRC; modified Lugano 2014 criteria) was 75.0% (95% confidence interval [CI], 61.3% to 85.8%). Three-year investigator-assessed PFS and overall survival rates were 80.9% (95% CI, 63.9% to 90.5%) and 89.3% (95% CI, 73.9% to 95.9%), respectively. At baseline, 21/40 patients had circulating lymphoma-specific clonotypes and underwent repeat testing at end of induction; all were minimal residual disease negative (10-5 sensitivity), with 16 (76.2%) CRs, 3 (14.3%) partial responses, and 2 (9.5%) with stable disease (IRC assessed). Grade 5 (fatal) adverse events (AEs) were reported in 5 patients. The efficacy of atezo-G-bendamustine in previously untreated FL did not appear superior to G-bendamustine efficacy as seen in the GALLIUM trial, and the addition of atezo to G-bendamustine was associated with an increased risk of AEs. Particularly due to the unfavorable safety profile, this regimen cannot be recommended in patients with previously untreated FL. This trial was registered at www.clinicaltrials.gov as #NCT02596971.
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Protocolos de Quimioterapia Combinada Antineoplásica , Linfoma Folicular , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Clorhidrato de Bendamustina/efectos adversos , Galio/uso terapéutico , Linfoma Folicular/tratamiento farmacológico , Rituximab/uso terapéutico , Anticuerpos Monoclonales Humanizados/efectos adversosRESUMEN
BACKGROUND: The combination of atezolizumab, a monoclonal antibody that targets programmed death-ligand 1 (PD-L1) and inhibits the interaction between PD-L1 and its receptors, and tazemetostat, an EZH2 inhibitor, may lead to selective epigenetic reprogramming, alter the tumor microenvironment, and provide additive or synergistic response to patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). MATERIALS AND METHODS: This was an open-label, phase Ib study assessing the safety, tolerability, and preliminary efficacy of atezolizumab plustazemetostat in patients with R/R DLBCL. Atezolizumab (1200 mg) was administered via intravenous (IV) infusion on day 1 of each cycle and tazemetostat (800 mg) was given orally twice daily (BID) on days 1 to 21. Primary endpoints were safety and tolerability, and to identify a recommended phase II dose (RP2D) for atezolizumab. Secondary efficacy endpoints included response rate and duration of response. RESULTS: A total of 43 patients were enrolled, receiving a median of 3 prior lines of treatment (range: 1-9). The RP2D for atezolizumab was 1200 mg IV infusion every 3 weeks in combination with tazemetostat 800 mg BID. At the RP2D, adverse events reported in ≥20% patients were anemia(11 patients [26%]), fatigue (10 patients [23%]), and nausea (10 patients [23%]). Overall response rate was 16% (complete response rate: 7%). Median progression-free survival was 2 months (range: 0-24) and median overall survival was 13 months (range: 1-29). CONCLUSIONS: The combination of atezolizumab and tazemetostat was determined to be safe and tolerable. However, anti-tumor activity of the combination was modest.
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Linfoma de Células B Grandes Difuso , Linfoma no Hodgkin , Anticuerpos Monoclonales Humanizados , Antígeno B7-H1 , Benzamidas , Compuestos de Bifenilo , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patología , Linfoma no Hodgkin/tratamiento farmacológico , Morfolinas , Piridonas , Microambiente TumoralRESUMEN
BACKGROUND: This was an open-label, phase 1b study assessing the safety, tolerability, preliminary efficacy and pharmacokinetics of the combination of atezolizumab and obinutuzumab in patients with relapsed/refractory follicular lymphoma (FL) or diffuse large B-cell lymphoma (DLBCL). There is a mechanistic rationale suggesting that this combination may enhance recruitment of both innate and adaptive immunity and be effective against CD20+ B-cell malignancies. MATERIALS AND METHODS: The study consisted of a safety evaluation stage and an expansion stage. Patients received obinutuzumab 1000 mg intravenously (IV) in cycle (C) 1, obinutuzumab plus atezolizumab 1200 mg IV for C2-8, and atezolizumab only from C9. Primary endpoints were to identify a recommended phase 2 dose (RP2D) for atezolizumab, and safety and tolerability in the safety and expansion stages. RESULTS: A total of 49 patients were enrolled (FL, n = 26; DLBCL, n = 23), with a median of 2 prior lines of treatment. The RP2D for atezolizumab was 1200 mg IV every 3 weeks. Adverse events reported in ≥ 20% of patients were fatigue (15 patients [31%]), nausea (13 patients [27%]), cough, and diarrhea (10 patients [20%] each). Objective response rate was 54% in the FL cohort (complete response [CR] rate: 23%) and 17% in the DLBCL cohort (CR: 4%). Median progression-free survival was 9 months for FL and 3 months for DLBCL. Median overall survival was not estimable for FL and 9 months for DLBCL. CONCLUSION: The combination of obinutuzumab and atezolizumab was determined to be safe and tolerable, with no new toxicities observed.
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Linfoma Folicular , Linfoma de Células B Grandes Difuso , Anticuerpos Monoclonales Humanizados/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , HumanosRESUMEN
The antiapoptotic protein BCL2 plays critical roles in regulating lymphocyte development and immune responses, and has also been implicated in tumorigenesis and tumor survival. However, it is unknown whether BCL2 is critical for antitumor immune responses. We evaluated whether venetoclax, a selective small-molecule inhibitor of BCL2, would influence the antitumor activity of immune checkpoint inhibitors (ICI). We demonstrate in mouse syngeneic tumor models that venetoclax can augment the antitumor efficacy of ICIs accompanied by the increase of PD-1+ T effector memory cells. Venetoclax did not impair human T-cell function in response to antigen stimuli in vitro and did not antagonize T-cell activation induced by anti-PD-1. Furthermore, we demonstrate that the antiapoptotic family member BCL-XL provides a survival advantage in effector T cells following inhibition of BCL2. Taken together, these data provide evidence that venetoclax should be further explored in combination with ICIs for cancer therapy. SIGNIFICANCE: The antiapoptotic oncoprotein BCL2 plays critical roles in tumorigenesis, tumor survival, lymphocyte development, and immune system regulation. Here we demonstrate that venetoclax, the first FDA/European Medicines Agency-approved BCL2 inhibitor, unexpectedly can be combined preclinically with immune checkpoint inhibitors to enhance anticancer immunotherapy, warranting clinical evaluation of these combinations.This article is highlighted in the In This Issue feature, p. 1.
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Inhibidores de Puntos de Control Inmunológico , Linfocitos T , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Sulfonamidas/farmacologíaRESUMEN
BACKGROUNDPD-1 and PD-L1 have been studied interchangeably in the clinic as checkpoints to reinvigorate T cells in diverse tumor types. Data for biologic effects of checkpoint blockade in human premalignancy are limited.METHODSWe analyzed the immunologic effects of PD-L1 blockade in a clinical trial of atezolizumab in patients with asymptomatic multiple myeloma (AMM), a precursor to clinical malignancy. Genomic signatures of PD-L1 blockade in purified monocytes and T cells in vivo were also compared with those following PD-1 blockade in lung cancer patients. Effects of PD-L1 blockade on monocyte-derived DCs were analyzed to better understand its effects on myeloid antigen-presenting cells.RESULTSIn contrast to anti-PD-1 therapy, anti-PD-L1 therapy led to a distinct inflammatory signature in CD14+ monocytes and increase in myeloid-derived cytokines (e.g., IL-18) in vivo. Treatment of AMM patients with atezolizumab led to rapid activation and expansion of circulating myeloid cells, which persisted in the BM. Blockade of PD-L1 on purified monocyte-derived DCs led to rapid inflammasome activation and synergized with CD40L-driven DC maturation, leading to greater antigen-specific T cell expansion.CONCLUSIONThese data show that PD-L1 blockade leads to distinct systemic immunologic effects compared with PD-1 blockade in vivo in humans, particularly manifest as rapid myeloid activation. These findings also suggest an additional role for PD-L1 as a checkpoint for regulating inflammatory phenotype of myeloid cells and antigen presentation in DCs, which may be harnessed to improve PD-L1-based combination therapies.TRIAL REGISTRATIONNCT02784483.FUNDINGThis work is supported, in part, by funds from NIH/NCI (NCI CA197603, CA238471, and CA208328).
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígeno B7-H1/inmunología , Mieloma Múltiple/tratamiento farmacológico , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Células Presentadoras de Antígenos/inmunología , Humanos , Inmunoterapia/métodos , Inflamación/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Mieloma Múltiple/inmunología , Receptor de Muerte Celular Programada 1/efectos de los fármacosRESUMEN
Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.
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Células Precursoras Eritroides/metabolismo , Hematopoyesis/genética , Telomerasa/metabolismo , Acortamiento del Telómero/genética , Telómero , Anemia Aplásica/genética , Anemia Aplásica/metabolismo , Animales , Proliferación Celular/genética , Daño del ADN , Ratones , Ratones Noqueados , Telomerasa/genéticaRESUMEN
The ability of a cell to undergo malignant transformation is both associated with and dependent on a concomitant increase in protein synthesis due to increased cell division rates and biosynthetic activities. Protein synthesis, in turn, depends upon the synthesis of ribosomes and thus ultimately on the transcription of ribosomal RNA by RNA polymerase I that occurs in the nucleolus. Enlargement of nucleoli has long been considered a hallmark of the malignant cell, but it is only recently that the rate of synthesis of rRNA in the nucleolus has been recognized as both a critical regulator of cellular proliferation and a potential target for therapeutic intervention. As might be expected, the factors regulating rRNA synthesis are both numerous and complex. It is the objective of this review to highlight recent advances in understanding how rRNA synthesis is perturbed in transformed mammalian cells and to consider the impact of these findings on the development of new approaches to the treatment of malignancies. In-depth analysis of the process of rRNA transcription itself may be found in several recently published reviews (Drygin et al., 2010, Annu Rev Pharmacol Toxicol 50:131-156; Bywater et al., 2013,Cancer Cell 22: 51-65; Hein et al., 2013,Trends Mol Med 19:643-654).
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Expresión Génica/fisiología , Ribosomas/metabolismo , Transcripción Genética/genética , Animales , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
The transcription initiation factor I (TIF-IA) is an important regulator of the synthesis of ribosomal RNA (rRNA) through its facilitation of the recruitment of RNA polymerase I (Pol I) to the ribosomal DNA promoter. Activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, which occurs commonly in acute myelogenous leukemia, enhances rRNA synthesis through TIF-IA stabilization and phosphorylation. We have discovered that TIF-IA coexists with a splicing isoform, TIF-90, which is expressed preferentially in the nucleolus and at higher levels in proliferating and transformed hematopoietic cells. TIF-90 interacts directly with Pol I to increase rRNA synthesis as a consequence of Akt activation. Furthermore, TIF-90 binds preferentially to a 90-kDa cleavage product of the actin binding protein filamin A (FLNA) that inhibits rRNA synthesis. Increased expression of TIF-90 overcomes the inhibitory effect of this cleavage product and stimulates rRNA synthesis. Because activated Akt also reduces FLNA cleavage, these results indicate that activated Akt and TIF-90 function in parallel to increase rRNA synthesis and, as a consequence, cell proliferation in leukemic cells. These results provide evidence that the direct targeting of Akt would be an effective therapy in acute leukemias in which Akt is activated.
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Empalme Alternativo , Proteínas Reguladoras de la Apoptosis/metabolismo , Filaminas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Western Blotting , Núcleo Celular/genética , Proliferación Celular , Inmunoprecipitación de Cromatina , Filaminas/genética , Humanos , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Ribosómico/antagonistas & inhibidores , ARN Ribosómico/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Células Tumorales CultivadasRESUMEN
We previously reported a rare germline variant (c.1-6531) that resulted in allele-specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5' upstream regulatory region, within distinct exons or in the 3'-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.
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Alelos , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Metilación de ADN , Ensayos Analíticos de Alto Rendimiento/métodos , Leucemia Linfocítica Crónica de Células B/metabolismo , Factores de Edad , Proteínas Reguladoras de la Apoptosis/genética , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Estudios de Cohortes , Cartilla de ADN/genética , Proteínas Quinasas Asociadas a Muerte Celular , Mutación de Línea Germinal/genética , Alemania , Humanos , Datos de Secuencia Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis. The DNA-hypomethylating agents 5-azacytidine and 5-aza-2'-deoxycytidine are effective treatments for patients with MDS, increasing the time to progression to acute myelogenous leukemia and improving overall response rates. Although genome-wide increases in DNA methylation have been documented in BM cells from MDS patients, the methylation signatures of specific gene promoters have not been correlated with the clinical response to these therapies. Recently, attention has been drawn to the potential etiologic role of decreased expression of specific ribosomal proteins in MDS and in other BM failure states. Therefore, we investigated whether rRNA expression is dysregulated in MDS. We found significantly decreased rRNA expression and increased rDNA promoter methylation in CD34(+) hematopoietic progenitor cells from the majority of MDS patients compared with normal controls. Treatment of myeloid cell lines with 5-aza-2'-deoxycytidine resulted in a significant decrease in the methylation of the rDNA promoter and an increase in rRNA levels. These observations suggest that an increase in rDNA promoter methylation can result in decreased rRNA synthesis that may contribute to defective hematopoiesis and BM failure in some patients with MDS.
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Metilación de ADN , ADN Ribosómico/genética , Síndromes Mielodisplásicos/genética , Regiones Promotoras Genéticas/genética , ARN Ribosómico/genética , Antígenos CD34/metabolismo , Línea Celular , Línea Celular Tumoral , Islas de CpG/genética , Citometría de Flujo , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaAsunto(s)
ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/uso terapéutico , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Dioxigenasas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Síndromes Mielodisplásicos/metabolismo , ARN Mensajero/metabolismo , Resultado del TratamientoRESUMEN
Epigenetic alterations, including gain or loss of DNA methylation, are a hallmark of nearly every malignancy. Changes in DNA methylation can impact expression of cancer-related genes including apoptosis regulators and tumor suppressors. Because such epigenetic changes are reversible, they are being aggressively investigated as potential therapeutic targets. Here we use the Emu-TCL1 transgenic mouse model of chronic lymphocytic leukemia (CLL) to determine the timing and patterns of aberrant DNA methylation, and to investigate the mechanisms that lead to aberrant DNA methylation. We show that CLL cells from Emu-TCL1 mice at various stages recapitulate epigenetic alterations seen in human CLL. Aberrant methylation of promoter sequences is observed as early as 3 months of age in these animals, well before disease onset. Abnormally methylated promoter regions include binding sites for the transcription factor FOXD3. We show that loss of Foxd3 expression due to an NF-kappaB p50/p50:HDAC1 repressor complex occurs in TCL1-positive B cells before methylation. Therefore, specific transcriptional repression is an early event leading to epigenetic silencing of target genes in murine and human CLL. These results provide strong rationale for the development of strategies to target NF-kappaB components in CLL and potentially other B-cell malignancies.
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Epigénesis Genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Animales , Metilación de ADN , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Factores de Transcripción Forkhead/metabolismo , Regulación Leucémica de la Expresión Génica , Silenciador del Gen , Histona Desacetilasa 1 , Histona Desacetilasas/metabolismo , Humanos , Ratones , Subunidad p50 de NF-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismoRESUMEN
Hypermethylation of CpG islands is a common epigenetic alteration associated with cancer. Global patterns of hypermethylation are tumor-type specific and nonrandom. The biological significance and the underlying mechanisms of tumor-specific aberrant promoter methylation remain unclear, but some evidence suggests that this specificity involves differential sequence susceptibilities, the targeting of DNA methylation activity to specific promoter sequences, or the selection of rare DNA methylation events during disease progression. Using restriction landmark genomic scanning on samples derived from tissue culture and in vivo models of T cell lymphomas, we found that MYC overexpression gave rise to a specific signature of CpG island hypermethylation. This signature reflected gene transcription profiles and was detected only in advanced stages of disease. The further inactivation of the Pten, p53, and E2f2 tumor suppressors in MYC-induced lymphomas resulted in distinct and diagnostic CpG island methylation signatures. Our data suggest that tumor-specific DNA methylation in lymphomas arises as a result of the selection of rare DNA methylation events during the course of tumor development. This selection appears to be driven by the genetic configuration of tumor cells, providing experimental evidence for a causal role of DNA hypermethylation in tumor progression and an explanation for the tremendous epigenetic heterogeneity observed in the evolution of human cancers. The ability to predict genome-wide epigenetic silencing based on relatively few genetic alterations will allow for a more complete classification of tumors and understanding of tumor cell biology.
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Islas de CpG/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Linfoma de Células T/genética , Animales , Células Cultivadas , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , ADN de Neoplasias/metabolismo , Modelos Animales de Enfermedad , Embrión de Mamíferos , Epigénesis Genética , Fibroblastos/metabolismo , Silenciador del Gen , Genes Supresores de Tumor , Humanos , Linfoma de Células T/metabolismo , Ratones , Ratones Desnudos , Ratones Transgénicos , Trasplante de Neoplasias , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , TransgenesRESUMEN
Molecular profiling may lead to a better understanding of a disease. This knowledge is especially important in malignancies, where multiple alterations are required during the progression from premalignant to malignant stages. Such information can be useful for the development of novel biomarkers that allow the prediction of a clinical course, response to treatment or early detection. Molecular data is also utilized to develop targeted therapies. Moreover, gene defects identified in profiling studies will help to understand the molecular pathways disrupted in the disease. This review provides an overview of molecular profiling approaches in chronic lymphocytic leukaemia (CLL). We will describe our current understanding of genetic alterations in CLL, the use of familial CLL for the identification of predisposing mutations, and the search for epigenetic alterations in CLL.
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Predisposición Genética a la Enfermedad , Leucemia Linfocítica Crónica de Células B/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Asociadas a Muerte Celular , Epigénesis Genética , Perfilación de la Expresión Génica/métodos , Silenciador del Gen , Genómica , HumanosRESUMEN
The heritability of B cell chronic lymphocytic leukemia (CLL) is relatively high; however, no predisposing mutation has been convincingly identified. We show that loss or reduced expression of death-associated protein kinase 1 (DAPK1) underlies cases of heritable predisposition to CLL and the majority of sporadic CLL. Epigenetic silencing of DAPK1 by promoter methylation occurs in almost all sporadic CLL cases. Furthermore, we defined a disease haplotype, which segregates with the CLL phenotype in a large family. DAPK1 expression of the CLL allele is downregulated by 75% in germline cells due to increased HOXB7 binding. In the blood cells from affected family members, promoter methylation results in additional loss of DAPK1 expression. Thus, reduced expression of DAPK1 can result from germline predisposition, as well as epigenetic or somatic events causing or contributing to the CLL phenotype.
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Proteínas Reguladoras de la Apoptosis/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Regulación hacia Abajo , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Desequilibrio Alélico , Animales , Apoptosis , Linfocitos B/citología , Cromosomas Humanos Par 9 , Metilación de ADN , Análisis Mutacional de ADN , Proteínas Quinasas Asociadas a Muerte Celular , Epigénesis Genética , Femenino , Mutación de Línea Germinal , Proteínas de Homeodominio/metabolismo , Humanos , Células Híbridas , Células Jurkat , Masculino , Ratones , Linaje , Mutación Puntual , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Enormous evidence has accumulated in the past decades that establishes the importance of epigenetic modifications in cancer and has resulted in shifting the focus from entirely genetic-based studies to integrated studies involving both genetic and epigenetic alterations. Chronic lymphocytic leukemia (CLL) is one such example where studies involving epigenetic aberrations have accelerated the search for affected genes, which was initially restricted to commonly deleted chromosomal regions. Many novel genes that are epigenetically silenced in CLL have been identified. Advances in the understanding of post-translational histone modifications and DNA methylation in normal and in CLL cells have proven to be extremely beneficial in finding powerful diagnostic markers, as well as in exploring novel therapies. At present, the field of epigenetics is at an evolving stage, but there is no doubt that further unraveling of its cause and effects in transformed cells will bring a new revolution in cancer therapeutics.
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Epigénesis Genética , Leucemia Linfocítica Crónica de Células B/genética , Animales , Metilación de ADN , Silenciador del Gen , HumanosRESUMEN
DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation. However, accurate and reproducible quantification of DNA methylation remains challenging. In this report, we describe Bio-COBRA (combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform), as a novel approach to quantitative DNA methylation analysis. The combination of a well-established method, COBRA, which interrogates DNA methylation via the restriction enzyme analysis of PCR-amplified bisulfite treated DNAs, with the Bioanalyzer platform allows for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. The sensitivity and reproducibility of Bio-COBRA make it a valuable tool for the analysis of DNA methylation in clinical samples, which could aid in the development of diagnostic and prognostic parameters with respect to disease detection and management.
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Islas de CpG , Metilación de ADN , Enzimas de Restricción del ADN , Electroforesis por Microchip/instrumentación , Reacción en Cadena de la Polimerasa/métodos , Sulfitos , Azacitidina/análogos & derivados , Azacitidina/farmacología , Línea Celular Tumoral , Decitabina , Humanos , Técnicas de Diagnóstico Molecular , Neoplasias/diagnóstico , Reproducibilidad de los ResultadosRESUMEN
B-cell chronic lymphocytic leukemia (CLL) is characterized by a clonal accumulation of mature neoplastic B cells indicating disruption of apoptosis. Restriction Landmark Genome Scanning was done to identify novel target genes silenced by CpG island methylation in CLL. Secreted frizzled-related protein 4 (SFRP4), a negative regulator of the Wnt signaling pathway, was found to be frequently methylated in CLL samples. Wnt signaling has been shown to control normal apoptotic behavior and is required for normal B-cell development whereas aberrant activation of this pathway has been observed in CLL. We show aberrant DNA methylation and silencing of SFRP4, as well as of additional SFRP family members, in primary CLL samples. Induction of their expression in a dose-dependent manner following treatment with a demethylating agent, 5-aza-2'-deoxycytidine, was shown. Of the five SFRP family members studied in detail, SFRP1 was hypermethylated and down-regulated in all CLL patient samples studied, suggesting that this epigenetic event is a critical step during leukemogenesis. Our results suggest that silencing of SFRPs by CpG island methylation is one possible mechanism contributing to aberrant activation of Wnt signaling pathway in CLL.