RESUMO
As cancer immunotherapies continue to expand across all areas of oncology, it is imperative to establish a standardized approach for defining and capturing clinically important toxicities, such as cytokine release syndrome (CRS). In this paper, we provide considerations for categorizing the variety of adverse events that may accompany CRS and for recognizing that presentations of CRS may differ among various immunotherapies (e.g., monoclonal antibodies, CAR T cell therapies and T cell engagers, which can include bispecific antibodies and other constructs). The goals of this paper are to ensure accurate and consistent identification of CRS in patients receiving immunotherapies in clinical studies to aid in reporting; enable more precise evaluation of the therapeutic risk-benefit profile and cross-study analyses; support evidence-based monitoring and management of important toxicities related to cancer immunotherapies; and improve patient care and outcomes. These efforts will become more important as the number and variety of molecular targets for immunotherapies broaden and as therapies with novel mechanisms continue to be developed.
Assuntos
Síndrome da Liberação de Citocina , Imunoterapia , Neoplasias , Anticorpos Biespecíficos , Ensaios Clínicos como Assunto , Síndrome da Liberação de Citocina/etiologia , Humanos , Imunoterapia/efeitos adversos , Imunoterapia Adotiva/efeitos adversos , Neoplasias/terapiaRESUMO
The phosphorylation of histone variant H2AX at DNA double-strand breaks is believed to be critical for recognition and repair of DNA damage. However, little is known about the molecular mechanism regulating the exchange of variant H2AX with conventional H2A in the context of the nucleosome. Here, we isolate the H2AX-associated factors, which include FACT (Spt16/SSRP1), DNA-PK, and PARP1 from a human cell line. Our analyses demonstrate that the H2AX-associated factors efficiently promote both integration and dissociation of H2AX and this exchange reaction is mainly catalyzed by FACT among the purified factors. The phosphorylation of H2AX by DNA-PK facilitates the exchange of nucleosomal H2AX by inducing conformational changes of the nucleosome. In contrast, poly-ADP-ribosylation of Spt16 by PARP1 significantly inhibits FACT activities for H2AX exchange. Thus, these data establish FACT as the major regulator involved in H2AX exchange process that is modulated by H2AX phosphorylation and Spt16 ADP-ribosylation.
Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Dimerização , Células HeLa , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/isolamento & purificação , Histonas/genética , Humanos , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Isoformas de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/isolamento & purificaçãoRESUMO
BACKGROUND: Thrombocytopenia in patients with myelodysplastic syndrome (MDS) is associated with shortened survival and an increased risk of evolution to acute myeloid leukemia (AML). In this study, the authors evaluated the efficacy of romiplostim in patients who had thrombocytopenia with low-risk/intermediate-1-risk MDS. METHODS: Patients who had thrombocytopenia with low-risk/intermediate-1-risk MDS (N = 250) were randomized 2:1 to receive romiplostim or placebo weekly for 58 weeks. RESULTS: The primary endpoint- the number of clinically significant bleeding events (CSBEs) per patient-had a hazard ratio for romiplostim:placebo of 0.83 (95% confidence interval, 0.66-1.05; P = .13). CSBEs were reduced significantly in the romiplostim group for patients who had baseline platelet counts ≥20 × 10(9) /L (P < .0001). For patients who had baseline platelet counts <20 × 10(9) /L, there was no difference in the number of CSBEs, but the platelet transfusion rates were higher in the placebo group (P < .0001), which may have affected the overall CSBE results in this group with severe thrombocytopenia. The incidence of bleeding events was reduced significantly in the romiplostim group (relative risk, 0.92), as were protocol-defined platelet transfusions (relative risk, 0.77). Platelet response rates according to 2006 International Working Group criteria were higher for the group that received romiplostim (odds ratio, 15.6). On the basis of interim data, an independent data monitoring committee advised halting study drug because of concerns regarding excess blasts and AML rates with romiplostim (interim hazard ratio, 2.51). At 58 weeks, the AML rates were 6% in the romiplostim group and 4.9% in the placebo group (hazard ratio, 1.20; 95% confidence interval, 0.38-3.84), and the overall survival rates were similar. CONCLUSIONS: Romiplostim treatment in patients with low-risk/intermediate-1-risk MDS increased platelet counts and decreased the number of bleeding events and platelet transfusions. Although study drug was discontinued because of an initial concern of AML risk, survival and AML rates were similar with romiplostim and placebo.
Assuntos
Síndromes Mielodisplásicas/tratamento farmacológico , Receptores Fc/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Trombocitopenia/tratamento farmacológico , Trombopoetina/uso terapêutico , Idoso , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/patologia , Placebos , Análise de Sobrevida , Trombocitopenia/patologia , Resultado do TratamentoRESUMO
Low endogenous erythropoietin levels and limited red blood cell transfusion history can predict response to erythropoiesis-stimulating agents in anaemic patients with myelodysplastic syndromes (MDS). The relationship between endogenous thrombopoietin (THPO) levels and platelet response to romiplostim is unknown. Variables including baseline endogenous THPO levels, transfusion needs, and platelet response were analysed in a randomized trial of 250 thrombocytopenic, lower-risk MDS patients (International Prognostic Scoring System low/intermediate-1). A predictive scoring system was developed based on log-likelihood ratios and logistic coefficients. Patients with HI-P (haematological improvement - platelets) responses had lower mean baseline THPO levels (P = 0·0497) and were more likely to have <6 platelet units transfused in the past year (P = 0·0027), as did patients with platelet responses ≥50% of weeks on romiplostim (P = 0·001 and P = 0·0037, respectively). A model for predicting response to romiplostim was developed and validated in a separate MDS cohort (N = 72). Patients in low-, intermediate-, and high-response groups had response rates of 17·4%, 29·6%, and 50·7%, respectively, for HI-P, and 17·4%, 33·8%, and 65·2%, respectively, for ≥50% response. For thrombocytopenic patients with lower-risk MDS, lower baseline THPO levels (<500 pg/ml) and limited platelet transfusion history predicted a greater likelihood of a subsequent platelet response to romiplostim.
Assuntos
Hematínicos/farmacologia , Modelos Biológicos , Síndromes Mielodisplásicas/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Trombocitopenia/tratamento farmacológico , Trombopoese/efeitos dos fármacos , Trombopoetina/farmacologia , Idoso , Transfusão de Sangue , Terapia Combinada , Feminino , Hematínicos/uso terapêutico , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/terapia , Contagem de Plaquetas , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptores Fc/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Medição de Risco , Trombocitopenia/etiologia , Trombopoetina/sangue , Trombopoetina/uso terapêuticoRESUMO
DNA methyltransferases (DNMTs) play an important role in establishing and maintaining DNA methylation. Aberrant expression of DNMTs and their isoforms has been found in many types of cancer, and their contribution to aberrant DNA methylation has been proposed. Here, we generated HEK 293T cells stably transfected with each of 13 different DNMTs (DNMT1, two DNMT3A isoforms, nine DNMT3B isoforms and DNMT3L) and assessed the DNA methylation changes induced by each DNMT. We obtained DNA methylation profiles of DNA repetitive elements and 1505 CpG sites from 808 cancer-related genes. We found that DNMTs have specific and overlapping target sites and their DNA methylation target profiles are a reflection of the DNMT domains. By examining H3K4me3 and H3K27me3 modifications in the 808 gene promoter regions using promoter ChIP-on-chip analysis, we found that specific de novo DNA methylation target sites of DNMT3A1 are associated with H3K4me3 modification that are transcriptionally active, whereas the specific target sites of DNMT3B1 are associated with H3K27me3 modification that are transcriptionally inactive. Our data suggest that different DNMT domains are responsible for targeting DNA methylation to specific regions of the genome, and this targeting might be associated with histone modifications.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/química , Metilação de DNA , DNA Metiltransferase 3A , Genes Neoplásicos , Células HEK293 , Histonas/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Sequências Repetitivas de Ácido Nucleico , DNA Metiltransferase 3BRESUMO
It was recently shown that a large portion of the human transcriptome can originate from within repetitive elements, leading to ectopic expression of protein-coding genes. However the mechanism of transcriptional activation of repetitive elements has not been definitively elucidated. For the first time, we directly demonstrate that hypomethylation of retrotransposons can cause altered gene expression in humans. We also reveal that active LINE-1s switch from a tetranucleosome to dinucleosome structure, acquiring H2A.Z- and nucleosome-free regions upstream of TSSs, previously shown only at active single-copy genes. Hypomethylation of a specific LINE-1 promoter was also found to induce an alternate transcript of the MET oncogene in bladder tumors and across the entire urothelium of tumor-bearing bladders. These data show that, in addition to contributing to chromosomal instability, hypomethylation of LINE-1s can alter the functional transcriptome and plays a role not only in human disease but also in disease predisposition.
Assuntos
Elementos Nucleotídeos Longos e Dispersos/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-met/genética , Receptores de Fatores de Crescimento/genética , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Metilação de DNA/genética , Humanos , Pessoa de Meia-Idade , Oncogenes , Ativação TranscricionalRESUMO
BACKGROUND: Retrotransposons have been extensively studied in plants and animals and have been shown to have an impact on human genome dynamics and evolution. Their ability to move within genomes gives retrotransposons to affect genome instability. METHODS: we examined the polymorphic inserted AluYa5, evolutionary young Alu, in the progesterone receptor gene to determine the effects of Alu insertion on molecular environment. We used mono-allelic inserted cell lines which carry both Alu-present and Alu-absent alleles. To determine the epigenetic change and gene expression, we performed restriction enzyme digestion, Pyrosequencing, and Chromatin Immunoprecipitation. RESULTS: We observed that the polymorphic insertion of evolutionally young Alu causes increasing levels of DNA methylation in the surrounding genomic area and generates inactive histone tail modifications. Consequently the Alu insertion deleteriously inactivates the neighboring gene expression. CONCLUSION: The mono-allelic Alu insertion cell line clearly showed that polymorphic inserted repetitive elements cause the inactivation of neighboring gene expression, bringing aberrant epigenetic changes.
Assuntos
Elementos Alu/genética , Metilação de DNA/genética , Genoma Humano , Transcrição Gênica , Alelos , Linhagem Celular , Epigênese Genética/genética , Evolução Molecular , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Receptores de Progesterona/genéticaRESUMO
DNA methylation is known to be associated with cell differentiation, aging, disease and cancer. There exists an expanding base of knowledge regarding tissue-specific DNA methylation, but we have little information about person-specific DNA methylation. Here, we analyze the DNA methylation patterns of multiple tissues from multiple individuals using a high-throughput quantitative assay of genome-wide DNA methylation, namely the Illumina GoldenGate BeadArray. DNA methylation patterns were largely conserved across 11 different tissues (r = 0.852) and across six individuals (r = 0.829), and we found that DNA was highly methylated in non-CpG islands and/or CpG sites that are not occupied by either H3K4me3 or H3K27me3 (P < 0.05). Finally, we found that the Illumina GoldenGate assay features a large number of probes (265/1505 probes, 17.6%) that contain single-nucleotide polymorphisms, which may interfere with DNA methylation analyses in genome-wide studies.
Assuntos
Ilhas de CpG , Metilação de DNA , Epigênese Genética , Autopsia , Estudo de Associação Genômica Ampla/métodos , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Sequential administration of DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors has demonstrated clinical efficacy in patients with hematologic malignancies. However, the mechanism behind their clinical efficacy remains controversial. In this study, the methylation dynamics of 4 TSGs (p15(INK4B), CDH-1, DAPK-1, and SOCS-1) were studied in sequential bone marrow samples from 30 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) who completed a minimum of 4 cycles of therapy with 5-azacytidine and entinostat. Reversal of promoter methylation after therapy was observed in both clinical responders and nonresponders across all genes. There was no association between clinical response and either baseline methylation or methylation reversal in the bone marrow or purified CD34(+) population, nor was there an association with change in gene expression. Transient global hypomethylation was observed in samples after treatment but was not associated with clinical response. Induction of histone H3/H4 acetylation and the DNA damage-associated variant histone gamma-H2AX was observed in peripheral blood samples across all dose cohorts. In conclusion, methylation reversal of candidate TSGs during cycle 1 of therapy was not predictive of clinical response to combination "epigenetic" therapy. This trial is registered with http://www.clinicaltrials.gov under NCT00101179.
Assuntos
Azacitidina/administração & dosagem , Benzamidas/administração & dosagem , Dano ao DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/tratamento farmacológico , Piridinas/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Análise Citogenética , Dano ao DNA/fisiologia , Esquema de Medicação , Epigênese Genética/fisiologia , Feminino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de TempoRESUMO
Hypomethylation of DNA repetitive elements is a common finding in cancer, but very little is known about the DNA methylation changes of different types of DNA repetitive elements, such as interspersed repeats (LINE1 and Alu Yb8) and tandem repeats (Sat-alpha, NBL-2 and D4Z4). We used bisulfite-PCR Pyrosequencing to quantitatively measure the DNA methylation of five different DNA repetitive elements in normal tissue and cancer. In all we studied 10 different tissues from four individuals undergoing autopsy, 34 paired normal and tumor tissues from patients with bladder cancer, 58 patients with chronic myelogenous leukemia and 23 patients with acute promyelocytic leukemia. We found that the DNA methylation of interspersed repeats (LINE1 and Alu Yb8) was very consistent from person to person and tissue to tissue while tandem DNA repeats appeared more variable in normal tissues. In bladder cancer we found clear hypomethylation of LINE1, Alu Yb8, Sat-alpha and NBL-2. Conversely, we found an increase in the DNA methylation levels of D4Z4 from normal to cancer. In contrast leukemia showed no significant changes in the DNA methylation of LINE1 and Alu Yb8, but DNA methylation increases in NBL-2 and D4Z4 tandem repeats. Our findings show that the changes in DNA methylation levels of individual DNA repetitive elements are unique for each repetitive element, which may reflect distinct epigenetic factors and may have important implications in the use of DNA methylation of repetitive elements as global DNA methylation biomarkers.
Assuntos
Metilação de DNA , Neoplasias/genética , Elementos Nucleotídeos Curtos e Dispersos , Sequências de Repetição em Tandem , Elementos Alu/genética , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Promielocítica Aguda/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Elementos Nucleotídeos Curtos e Dispersos/genética , Neoplasias da Bexiga Urinária/genéticaRESUMO
Aberrant DNA methylation patterns, including global hypomethylation, gene-specific hypermethylation/hypomethylation, and loss of imprinting (LOI), are common in acute myelogenous leukemia (AML) and other cancer tissues. We investigated for the first time whether such epigenetic changes are induced in healthy subjects by low-level exposure to benzene, a widespread pollutant associated with AML risk. Blood DNA samples and exposure data were obtained from subjects with different levels of benzene exposure, including 78 gas station attendants, 77 traffic police officers, and 58 unexposed referents in Milan, Italy (personal airborne benzene range, < 6-478 microg/m(3)). Bisulfite-PCR pyrosequencing was used to quantitate DNA methylation in long interspersed nuclear element-1 (LINE-1) and AluI repetitive elements as a surrogate of genome-wide methylation and examine gene-specific methylation of MAGE-1 and p15. Allele-specific pyrosequencing of the H19 gene was used to detect LOI in 96 subjects heterozygous for the H19 imprinting center G/A single-nucleotide polymorphism. Airborne benzene was associated with a significant reduction in LINE-1 (-2.33% for a 10-fold increase in airborne benzene levels; P = 0.009) and AluI (-1.00%; P = 0.027) methylation. Hypermethylation in p15 (+0.35%; P = 0.018) and hypomethylation in MAGE-1 (-0.49%; P = 0.049) were associated with increasing airborne benzene levels. LOI was found only in exposed subjects (4 of 73, 5.5%) and not in referents (0 of 23, 0.0%). However, LOI was not significantly associated with airborne benzene (P > 0.20). This is the first human study to link altered DNA methylation, reproducing the aberrant epigenetic patterns found in malignant cells, to low-level carcinogen exposure.
Assuntos
Benzeno/intoxicação , Metilação de DNA/efeitos dos fármacos , Exposição Ocupacional , Adulto , Idoso , Antígenos de Neoplasias/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , DNA/sangue , Relação Dose-Resposta a Droga , Exposição Ambiental , Feminino , Impressão Genômica/efeitos dos fármacos , Humanos , Exposição por Inalação , Elementos Nucleotídeos Longos e Dispersos/efeitos dos fármacos , Masculino , Antígenos Específicos de Melanoma , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , RNA Longo não Codificante , RNA não Traduzido/efeitos dos fármacos , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
2'-Deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine (NPEOC-DAC), decitabine with a modification of the N4 position of the azacitidine ring can be used to inhibit DNA methyltransferase. This modification protects the azacitidine ring and can be cleaved by carboxylesterase to release decitabine. NPEOC-DAC was 23-fold less potent at low doses (<10microM) than decitabine at inhibiting DNA methylation, and was also associated with a 3-day delay in its effect. However, at doses > or = 10microM NPEOC-DAC was more effective at inhibiting DNA methylation. Theses differences between decitabine and NPEOC-DAC are dependent on the cleavage of the carboxylester bond, and could be potentially exploited pharmacologically.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Hidrolases de Éster Carboxílico/metabolismo , DNA-Citosina Metilases/antagonistas & inibidores , Pró-Fármacos/farmacologia , Antimetabólitos Antineoplásicos/química , Azacitidina/antagonistas & inibidores , Azacitidina/química , Azacitidina/farmacologia , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Decitabina , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Humanos , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Pró-Fármacos/química , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
PURPOSE: 1-beta-D-Arabinofuranosylcytosine (cytarabine; ara-C) is the most active agent in myeloid leukemia. 5-Aza-2'-deoxycytidine (DAC) is a cytosine analogue that inhibits DNA methylation and also has activity in myeloid leukemia. Therefore, we investigated combining these two drugs in human leukemia cell lines in vitro. EXPERIMENTAL DESIGN: We initially examined the effects of ara-C and DAC on human leukemia cell lines HL60, ML-1, RAji, and Jurkat. We measured IC(50) of DAC and ara-C in these cell lines and calculated a combination index of these two drugs given either simultaneously or sequentially. In searching for mechanisms relative to epigenetic regulation for this effect, we examined DNA methylation of LINE and Alu repetitive elements as a surrogate for global genomic DNA methylation. In addition, we sorted Annexin V positive and negative cells and measured differences in LINE methylation between them. RESULTS: The combination of DAC and ara-C showed additive induction of cell death in ML-1 and synergistic induction in HL60, Raji, and Jurkat. Sequentially, DAC followed by ara-C was a synergistic combination in all cell lines. Low-dose DAC induced more hypomethylation than high doses of the drug, whereas ara-C had no effects on methylation. The combination of ara-C with DAC either together or DAC followed by ara-C resulted in inhibition of LINE demethylation in HL60. The RIL gene, which is silenced by DNA hypermethylation, was activated by DAC, but the addition of ara-C to DAC reduced RIL gene activation. DAC treatment increased H3 Lys(9) acetylation of Alu elements, whereas ara-C had no effect, and the addition of ara-C to DAC inhibited this effect. Finally, we showed that after DAC exposure, Annexin V positive cells were more hypomethylated than Annexin V negative cells. CONCLUSION: The combination of DAC and ara-C showed additive or synergistic effects on cell death in four human leukemia cell lines in vitro, but antagonism in terms of epigenetic effects. One possible explanation for these paradoxical observations is that hypomethylated cells are sensitized to cell killing by ara-C. These data suggest that DAC used in combination with ara-C has clinical potential in the treatment of acute myeloid leukemia.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Citarabina/farmacologia , Células HL-60/efeitos dos fármacos , Anexina A5/metabolismo , Azacitidina/farmacologia , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/genética , Decitabina , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Zebularine (1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) was studied as both a 2 '-deoxyribosyl 5 '-triphosphate derivative and as a template incorporated into an oligonucleotide. Using a novel pyrosequencing assay, zebularine acted as cytosine analog and was incorporated into DNA with a template pairing profile most similar to cytosine, pairing with greatest efficiency opposite guanine in the template strand. Guanine was incorporated with greater affinity than adenine opposite a zebularine in the template strand. Computer modeling of base-pairing structures supported a better fit of zebularine opposite guanine than adenine. Zebularine acts as a cytosine analog, which supports its activity as an inhibitor of cytosine methyltransferase.
Assuntos
Pareamento de Bases , Citidina/análogos & derivados , Inibidores Enzimáticos/química , Modelos Moleculares , Oligonucleotídeos/química , Citidina/química , DNA-Citosina Metilases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Oligonucleotídeos/síntese química , Análise de Sequência de DNA/métodosRESUMO
5-Aza-2'-deoxycytidine (decitabine) is postulated to have clinical activity in myeloid leukemias via its ability to inhibit DNA methylation. To study this, we examined DNA methylation in patients with leukemia treated with decitabine. Five days after the treatment, total genomic 5-methylcytosine/cytosine decreased on average by 14% (from 4.3% to 3.7%), whereas methylation of repetitive DNA elements showed a mean decrease of 9% and 16% for Alu and long interspersed nucleotide elements, respectively. Methylation decreased linearly with increasing doses between 5 and 20 mg/m(2)/d (r = 0.88; P = 0.05) but showed a plateau above that. Hypomethylation correlated with response in patients with acute myelogenous leukemia treated with low doses (5-20 mg/m(2)/d), but patients with chronic myelogenous leukemia treated with high doses (100-180 mg/m(2)/d) showed no such correlation. Aberrant methylation of p15 (>10%) was found in 27% of patients, and 80% of these showed a decrease by at least one third, but this did not correlate with response. The imprinted gene H19 showed little change in methylation after decitabine. In conclusion, we show dose-dependent hypomethylation after decitabine at low doses. Increasing the dose, which has been shown previously to result in a reduced response rate, was not accompanied by further hypomethylation.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Decitabina , Relação Dose-Resposta a Droga , Feminino , Genes p53/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , RNA Longo não Codificante , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
BACKGROUND: Cell division occurs during normal human development and aging. Despite the likely importance of cell division to human pathology, it has been difficult to infer somatic cell mitotic ages (total numbers of divisions since the zygote) because direct counting of lifetime numbers of divisions is currently impractical. Here we attempt to infer relative mitotic ages with a molecular clock hypothesis. Somatic genomes may record their mitotic ages because greater numbers of replication errors should accumulate after greater numbers of divisions. Mitotic ages will vary between cell types if they divide at different times and rates. METHODS: Age-related increases in DNA methylation at specific CpG sites (termed "epigenetic molecular clocks") have been previously observed in mitotic human epithelium like the intestines and endometrium. These CpG rich sequences or "tags" start unmethylated and potentially changes in methylation during development and aging represent replication errors. To help distinguish between mitotic versus time-associated changes, DNA methylation tag patterns at 8-20 CpGs within three different genes, two on autosomes and one on the X-chromosome were measured by bisulfite sequencing from heart, brain, kidney and liver of autopsies from 21 individuals of different ages. RESULTS: Levels of DNA methylation were significantly greater in adult compared to fetal or newborn tissues for two of the three examined tags. Consistent with the relative absence of cell division in these adult tissues, there were no significant increases in tag methylation after infancy. CONCLUSION: Many somatic methylation changes at certain CpG rich regions or tags appear to represent replication errors because this methylation increases with chronological age in mitotic epithelium but not in non-mitotic organs. Tag methylation accumulates differently in different tissues, consistent with their expected genealogies and mitotic ages. Although further studies are necessary, these results suggest numbers of divisions and ancestry are at least partially recorded by epigenetic replication errors within somatic cell genomes.
Assuntos
Envelhecimento/genética , Ilhas de CpG , Metilação de DNA , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/crescimento & desenvolvimento , Linhagem da Célula , Pré-Escolar , Epigênese Genética , Feminino , Coração/crescimento & desenvolvimento , Humanos , Lactente , Recém-Nascido , Rim/crescimento & desenvolvimento , Fígado/crescimento & desenvolvimento , Masculino , Pessoa de Meia-Idade , Especificidade de ÓrgãosRESUMO
Myelodysplastic syndromes (MDS) comprise a collection of hematologic disorders characterized by deficiencies in peripheral blood cells, particularly those of the granulocyte, erythrocyte, and megakaryocyte lineages. A number of chromosomal abnormalities are associated with the development of MDS, including mutations involving chromosomes 7q, 5q, 20q, 6q, 11q, and 13q. Until recently, supportive care or allogeneic stem cell transplantation (ASCT) were the only available treatment options for this disease. ASCT is potentially curative but poses high mortality risk in the predominantly elderly MDS population. Supportive care options traditionally included blood transfusions and antibiotics, with the goal of reducing morbidity from ineffective hematopoiesis and improving quality of life. This approach has been enhanced by treatment with various growth factors aimed at stimulating blood cell production, including erythropoietin and granulocyte colony-stimulating factor. These growth factors, along with the use of iron chelation therapy to counteract the iron overload that can develop after frequent transfusions, have recently been shown to have the potential to prolong survival in MDS patients. In addition to these advances in supportive care, three new agents have been approved for the treatment of MDS in the past 3 years. Lenalidomide, azacitidine, and decitabine all target molecular processes underlying the pathophysiology of MDS and have shown considerable activity and, more recently, potential survival benefits in various subgroups of patients. Considerable changes in the treatment of MDS are expected in coming years as these and other novel agents are tested alone and in combination with one another to further refine the treatment paradigm for MDS.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Fatores Imunológicos/uso terapêutico , Síndromes Mielodisplásicas/tratamento farmacológico , Azacitidina/análogos & derivados , Azacitidina/uso terapêutico , Ensaios Clínicos como Assunto , Decitabina , Epigênese Genética , Humanos , Lenalidomida , Análise de Sobrevida , Talidomida/análogos & derivados , Talidomida/uso terapêuticoRESUMO
Genomic instability, including genetic mutations and chromosomal rearrangements, can lead to cancer development. Aberrant DNA methylation occurs commonly in cancer cells. The aim of this study is to determine the effects of a specific chromosomal lesion the BCR-ABL translocation t(9:22), in establishing DNA methylation profiles in cancer. Materials and methods We compared DNA methylation of 1,505 selected promoter CpGs in chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL) with and without the Philadelphia chromosome t(9:22), CD34+ hematopoietic stem cells transfected with BCR-ABL, and other tumors without BCR-ABL (acute promyelocytic leukemia (APL) and gastrointestinal stromal tumors (GIST). In this study, the DNA methylation profile of CML was more closely related to APL, another myeloid leukemia, than Ph+ ALL. Although DNA methylation profiles were consistent within a specific tumor type, overall DNA methylation profiles were no influenced by BCR-ABL gene translocation in the cancers and tissues studied. We conclude that DNA methylation profiles may reflect the cell of origin in cancers rather than the chromosomal lesions involved in leukemogenesis.
RESUMO
Purpose: A phase I study was conducted to determine the MTD, dose-limiting toxicities (DLT), and pharmacokinetics of fenretinide delivered as an intravenous emulsion in relapsed/refractory hematologic malignancies.Experimental Design: Fenretinide (80-1,810 mg/m2/day) was administered by continuous infusion on days 1 to 5, in 21-day cycles, using an accelerated titration design.Results: Twenty-nine patients, treated with a median of three prior regimens (range, 1-7), were enrolled and received the test drug. Ninety-seven courses were completed. An MTD was reached at 1,280 mg/m2/day for 5 days. Course 1 DLTs included 6 patients with hypertriglyceridemia, 4 of whom were asymptomatic; 2 patients experienced DLT thrombocytopenia (asymptomatic). Of 11 patients with response-evaluable peripheral T-cell lymphomas, two had complete responses [CR, progression-free survival (PFS) 68+ months; unconfirmed CR, PFS 14+ months], two had unconfirmed partial responses (unconfirmed PR, PFS 5 months; unconfirmed PR, PFS 6 months), and five had stable disease (2-12 cycles). One patient with mature B-cell lymphoma had an unconfirmed PR sustained for two cycles. Steady-state plasma levels were approximately 10 mcg/mL (mid-20s µmol/L) at 640 mg/m2/day, approximately 14 mcg/mL (mid-30s µmol/L) at 905 mg/m2/day, and approximately 22 mcg/mL (mid-50s µmol/L) at 1,280 mg/m2/day.Conclusions: Intravenous fenretinide obtained significantly higher plasma levels than a previous capsule formulation, had acceptable toxicities, and evidenced antitumor activity in peripheral T-cell lymphomas. A recommended phase II dosing is 600 mg/m2 on day 1, followed by 1,200 mg/m2 on days 2 to 5, every 21 days. A registration-enabling phase II study in relapsed/refractory PTCL (ClinicalTrials.gov identifier: NCT02495415) is ongoing. Clin Cancer Res; 23(16); 4550-5. ©2017 AACR.
Assuntos
Fenretinida/uso terapêutico , Neoplasias Hematológicas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , California , Relação Dose-Resposta a Droga , Esquema de Medicação , Resistencia a Medicamentos Antineoplásicos , Feminino , Fenretinida/administração & dosagem , Fenretinida/farmacocinética , Neoplasias Hematológicas/patologia , Humanos , Infusões Intravenosas , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Indução de Remissão , Trombocitopenia/induzido quimicamente , Adulto JovemRESUMO
Several biological phenomena depend on differential methylation of chromosomal strands. While understanding the role of these processes requires information on allele-specific methylation, the available methodologies are not quantitative or labor-intensive. We describe a novel, rapid method to quantitate allele-specific DNA methylation based on the combination of bisulfite PCR and Pyrosequencing. In this method, DNA is first treated with sodium bisulfite, which converts cytosine but not 5-methylcytosine to uracil. Genes of interest are subsequently amplified using PCR. Allele-specific methylation can then be determined by pyrosequencing each allele individually using sequencing primers that incorporate single nucleotide polymorphisms (SNPs) that allow differentiation between the two parental alleles. This allele-specific methylation methodology can potentially afford quantitative analyses relevant to the regulation of X chromosome inactivation, allele-specific expression of genes in the immune system, repetitive elements, and genomic imprinting. As an illustration of our new method, we quantitated allele-specific methylation of the differentially methylated region of the H19 gene, which is imprinted. Although we could reliably determine allele-specific methylation with our technique, additional studies will be required to confirm the ability of our assay to measure loss of imprinting.