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1.
Mol Cancer ; 23(1): 114, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38811984

RESUMO

BACKGROUND: Prostate cancer develops through malignant transformation of the prostate epithelium in a stepwise, mutation-driven process. Although activator protein-1 transcription factors such as JUN have been implicated as potential oncogenic drivers, the molecular programs contributing to prostate cancer progression are not fully understood. METHODS: We analyzed JUN expression in clinical prostate cancer samples across different stages and investigated its functional role in a Pten-deficient mouse model. We performed histopathological examinations, transcriptomic analyses and explored the senescence-associated secretory phenotype in the tumor microenvironment. RESULTS: Elevated JUN levels characterized early-stage prostate cancer and predicted improved survival in human and murine samples. Immune-phenotyping of Pten-deficient prostates revealed high accumulation of tumor-infiltrating leukocytes, particularly innate immune cells, neutrophils and macrophages as well as high levels of STAT3 activation and IL-1ß production. Jun depletion in a Pten-deficient background prevented immune cell attraction which was accompanied by significant reduction of active STAT3 and IL-1ß and accelerated prostate tumor growth. Comparative transcriptome profiling of prostate epithelial cells revealed a senescence-associated gene signature, upregulation of pro-inflammatory processes involved in immune cell attraction and of chemokines such as IL-1ß, TNF-α, CCL3 and CCL8 in Pten-deficient prostates. Strikingly, JUN depletion reversed both the senescence-associated secretory phenotype and senescence-associated immune cell infiltration but had no impact on cell cycle arrest. As a result, JUN depletion in Pten-deficient prostates interfered with the senescence-associated immune clearance and accelerated tumor growth. CONCLUSIONS: Our results suggest that JUN acts as tumor-suppressor and decelerates the progression of prostate cancer by transcriptional regulation of senescence- and inflammation-associated genes. This study opens avenues for novel treatment strategies that could impede disease progression and improve patient outcomes.


Assuntos
Progressão da Doença , PTEN Fosfo-Hidrolase , Neoplasias da Próstata , Microambiente Tumoral , Masculino , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Animais , Camundongos , Humanos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Microambiente Tumoral/imunologia , Fenótipo Secretor Associado à Senescência , Proteínas Proto-Oncogênicas c-jun/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Senescência Celular/genética , Modelos Animais de Doenças
2.
Blood ; 138(25): 2670-2685, 2021 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33945616

RESUMO

Patients with chronic lymphocytic leukemia (CLL) bearing TP53 mutations experience chemorefractory disease and are therefore candidates for targeted therapy. However, the significance of low-burden TP53 mutations with <10% variant allele frequency (VAF) remains a matter for debate. Herein, we describe clonal evolution scenarios of low-burden TP53 mutations, the clinical impact of which we analyzed in a "real-world" CLL cohort. TP53 status was assessed by targeted next-generation sequencing (NGS) in 511 patients entering first-line treatment with chemo- and/or immunotherapy and 159 patients in relapse before treatment with targeted agents. Within the pretherapy cohort, 16% of patients carried low-burden TP53 mutations (0.1% to 10% VAF). Although their presence did not significantly shorten event-free survival after first-line therapy, it affected overall survival (OS). In a subgroup with TP53 mutations of 1% to 10% VAF, the impact on OS was observed only in patients with unmutated IGHV who had not received targeted therapy, as patients benefited from switching to targeted agents, regardless of initial TP53 mutational status. Analysis of the clonal evolution of low-burden TP53 mutations showed that the highest expansion rates were associated with fludarabine, cyclophosphamide, and rituximab regimen in both first- and second-line treatments (median VAF increase, 14.8× and 11.8×, respectively) in contrast to treatment with less intense treatment regimens (1.6×) and no treatment (0.8×). In the relapse cohort, 33% of patients carried low-burden TP53 mutations, which did not expand significantly upon targeted treatment (median VAF change, 1×). Sporadic cases of TP53 mutations' clonal shifts were connected with the development of resistance-associated mutations. Altogether, our data support the incorporation of low-burden TP53 variants in clinical decision making.


Assuntos
Evolução Clonal , Leucemia Linfocítica Crônica de Células B/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Evolução Clonal/efeitos dos fármacos , Feminino , Humanos , Imunoterapia , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/terapia , Masculino , Pessoa de Meia-Idade , Mutação/efeitos dos fármacos , Células Tumorais Cultivadas
3.
Mol Cancer ; 21(1): 89, 2022 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-35354467

RESUMO

BACKGROUND: Frequent truncation mutations of the histone lysine N-methyltransferase KMT2C have been detected by whole exome sequencing studies in various cancers, including malignancies of the prostate. However, the biological consequences of these alterations in prostate cancer have not yet been elucidated. METHODS: To investigate the functional effects of these mutations, we deleted the C-terminal catalytic core motif of Kmt2c specifically in mouse prostate epithelium. We analysed the effect of Kmt2c SET domain deletion in a Pten-deficient PCa mouse model in vivo and of truncation mutations of KMT2C in a large number of prostate cancer patients. RESULTS: We show here for the first time that impaired KMT2C methyltransferase activity drives proliferation and PIN formation and, when combined with loss of the tumour suppressor PTEN, triggers loss of senescence, metastatic dissemination and dramatically reduces life expectancy. In Kmt2c-mutated tumours we show enrichment of proliferative MYC gene signatures and loss of expression of the cell cycle repressor p16INK4A. In addition, we observe a striking reduction in disease-free survival of patients with KMT2C-mutated prostate cancer. CONCLUSIONS: We identified truncating events of KMT2C as drivers of proliferation and PIN formation. Loss of PTEN and KMT2C in prostate cancer results in loss of senescence, metastatic dissemination and reduced life expectancy. Our data demonstrate the prognostic significance of KMT2C mutation status in prostate cancer patients. Inhibition of the MYC signalling axis may be a viable treatment option for patients with KMT2C truncations and therefore poor prognosis.


Assuntos
Metiltransferases , Neoplasias da Próstata , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/fisiologia , Humanos , Masculino , Metiltransferases/genética , Camundongos , Mutação , Neoplasias da Próstata/metabolismo , Sequenciamento do Exoma
4.
Int J Cancer ; 148(3): 731-747, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33034050

RESUMO

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3'-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein µ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRß) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3- and androgen-mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth.


Assuntos
Cristalinas/genética , Cristalinas/metabolismo , Regulação para Baixo , Neoplasias da Próstata/patologia , Transdução de Sinais , Linhagem Celular Tumoral , Colina/administração & dosagem , Colina/análogos & derivados , Estudos de Coortes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metabolômica , Estadiamento de Neoplasias , Células PC-3 , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Prognóstico , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores dos Hormônios Tireóideos/genética , Análise de Sequência de RNA , Análise Serial de Tecidos , Tri-Iodotironina/antagonistas & inibidores , Tri-Iodotironina/metabolismo , Cristalinas mu
5.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33804860

RESUMO

The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A, has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson's disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.


Assuntos
Redes Reguladoras de Genes , Doenças Neurodegenerativas/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Linhagem Celular Tumoral , Éxons , Células HEK293 , Humanos , Neurônios/metabolismo , Motivos de Nucleotídeos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transcriptoma
6.
Nucleic Acids Res ; 46(2): 765-781, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29220521

RESUMO

RNA editing by targeted insertion and deletion of uridine is crucial to generate translatable mRNAs from the cryptogenes of the mitochondrial genome of kinetoplastids. This type of editing consists of a stepwise cascade of reactions generally proceeding from 3' to 5' on a transcript, resulting in a population of partially edited as well as pre-edited and completely edited molecules for each mitochondrial cryptogene of these protozoans. Often, the number of uridines inserted and deleted exceed the number of nucleotides that are genome-encoded. Thus, analysis of kinetoplastid mitochondrial transcriptomes has proven frustratingly complex. Here we present our analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, our new tool which allows comprehensive characterization of RNA editing, not relying on targeted transcript amplification and on prior knowledge of final edited products. T-Aligner implements a pipeline of read mapping, visualization of all editing states and their coverage, and assembly of canonical and alternative translatable mRNAs. We also assess T-Aligner functionality on a more challenging deep sequencing read input from Trypanosoma cruzi. The analysis reveals that transcripts of cryptogenes of both species undergo very complex editing that includes the formation of alternative open reading frames and whole categories of truncated editing products.


Assuntos
Mitocôndrias/genética , Edição de RNA , RNA Mitocondrial/genética , Trypanosomatina/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Genoma Mitocondrial/genética , Genoma de Protozoário/genética , Mitocôndrias/metabolismo , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , Splicing de RNA , RNA Mitocondrial/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosomatina/metabolismo
7.
Eur J Immunol ; 48(2): 273-282, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29080214

RESUMO

Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (IgκAID yy1f/f ) or that express normal AID levels (yy1f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sµ switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination.


Assuntos
Linfócitos B/fisiologia , Mutagênese/genética , Fator de Transcrição YY1/genética , Animais , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Feminino , Switching de Imunoglobulina/genética , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação/genética , Ligação Proteica , Hipermutação Somática de Imunoglobulina/genética , Ubiquitinação
8.
Bioinformatics ; 33(23): 3802-3804, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29036643

RESUMO

MOTIVATION: Sanger sequencing is still being employed for sequence variant detection by many laboratories, especially in a clinical setting. However, chromatogram interpretation often requires manual inspection and in some cases, considerable expertise. RESULTS: We present GLASS, a web-based Sanger sequence trace viewer, editor, aligner and variant caller, built to assist with the assessment of variations in 'curated' or user-provided genes. Critically, it produces a standardized variant output as recommended by the Human Genome Variation Society. AVAILABILITY AND IMPLEMENTATION: GLASS is freely available at http://bat.infspire.org/genomepd/glass/ with source code at https://github.com/infspiredBAT/GLASS. CONTACT: nikos.darzentas@gmail.com or malcikova.jitka@fnbrno.cz. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Técnicas de Genotipagem/métodos , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Software , Processamento Alternativo , Humanos , Polimorfismo Genético , Proteína Supressora de Tumor p53/genética
9.
Epilepsia ; 58(10): 1782-1793, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28815576

RESUMO

OBJECTIVE: Mesial temporal lobe epilepsy (mTLE) is a severe neurological disorder characterized by recurrent seizures. mTLE is frequently accompanied by neurodegeneration in the hippocampus resulting in hippocampal sclerosis (HS), the most common morphological correlate of drug resistance in mTLE patients. Incomplete knowledge of pathological changes in mTLE+HS complicates its therapy. The pathological mechanism underlying mTLE+HS may involve abnormal gene expression regulation, including posttranscriptional networks involving microRNAs (miRNAs). miRNA expression deregulation has been reported in various disorders, including epilepsy. However, the miRNA profile of mTLE+HS is not completely known and needs to be addressed. METHODS: Here, we have focused on hippocampal miRNA profiling in 33 mTLE+HS patients and nine postmortem controls to reveal abnormally expressed miRNAs. In this study, we significantly reduced technology-related bias (the most common source of false positivity in miRNA profiling data) by combining two different miRNA profiling methods, namely next generation sequencing and miRNA-specific quantitative real-time polymerase chain reaction. RESULTS: These methods combined have identified and validated 20 miRNAs with altered expression in the human epileptic hippocampus; 19 miRNAs were up-regulated and one down-regulated in mTLE+HS patients. Nine of these miRNAs have not been previously associated with epilepsy, and 19 aberrantly expressed miRNAs potentially regulate the targets and pathways linked with epilepsy (such as potassium channels, γ-aminobutyric acid, neurotrophin signaling, and axon guidance). SIGNIFICANCE: This study extends current knowledge of miRNA-mediated gene expression regulation in mTLE+HS by identifying miRNAs with altered expression in mTLE+HS, including nine novel abnormally expressed miRNAs and their putative targets. These observations further encourage the potential of microRNA-based biomarkers or therapies.


Assuntos
Epilepsia do Lobo Temporal/genética , Regulação da Expressão Gênica , Hipocampo/patologia , MicroRNAs/genética , Adolescente , Adulto , Simulação por Computador , Regulação para Baixo , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/patologia , Epilepsia do Lobo Temporal/cirurgia , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Hipocampo/metabolismo , Hipocampo/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Esclerose , Análise de Sequência de RNA , Regulação para Cima , Adulto Jovem
10.
Tumour Biol ; 36(5): 3371-80, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25527155

RESUMO

TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.


Assuntos
Genes p53 , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único
11.
Carcinogenesis ; 35(5): 992-1002, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24306027

RESUMO

MicroRNA (miRNA) expression is deregulated in many tumors including chronic lymphocytic leukemia (CLL). Although the particular mechanism(s) responsible for their aberrant expression is not well characterized, the presence of mutations and single-nucleotide polymorphisms (SNPs) in miRNA genes, possibly affecting their secondary structure and expression, has been described. In CLL; however, the impact and frequency of such variations have yet to be elucidated. Using a custom resequencing microarray, we screened sequence variations in 109 cancer-related pre-miRNAs in 98 CLL patients. Additionally, the primary regions of miR-29b-2/29c and miR-16-1 were analyzed by Sanger sequencing in another cohort of 213 and 193 CLL patients, respectively. Altogether, we describe six novel miR-sequence variations and the presence of SNPs (n = 27), most of which changed the miR-secondary structure. Moreover, some of the identified SNPs have a significantly different frequency in CLL when compared with a control population. Additionally, we identified a novel variation in miR-16-1 that had not been described previously in CLL patients. We show that this variation affects the expression of mature miR-16-1. We also show that the expression of another miRNA with pathogenetic relevance for CLL, namely miR-29b-2, is influenced by the presence of a polymorphic insertion, which is more frequent in CLL than in a control population. Altogether, these data suggest that sequence variations may occur during CLL development and/or progression.


Assuntos
Variação Genética , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Adulto , Idoso , Alelos , Aberrações Cromossômicas , Feminino , Regulação Leucêmica da Expressão Gênica , Frequência do Gene , Mutação em Linhagem Germinativa , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , MicroRNAs/química , Pessoa de Meia-Idade , Mutação , Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
12.
Blood ; 119(9): 2110-3, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22234685

RESUMO

MicroRNAs (miRNAs) play a key role in chronic lymphocytic leukemia as well as in normal B cells. Notably, miRNA gene encoding miR-650 and its homologs overlap with several variable (V) subgenes coding for lambda immunoglobulin (IgLλ). Recent studies describe the role of miR-650 in solid tumors, but its role in chronic lymphocytic leukemia (CLL) has not yet been studied. Our experiments demonstrate that miR-650 expression is regulated by coupled expression with its host gene for IgLλ. This coupling provides a unique yet unobserved mechanism for microRNA gene regulation. We determine that higher expression of miR-650 is associated with a favorable CLL prognosis and influences the proliferation capacity of B cells. We also establish that in B cells, miR-650 targets proteins important in cell proliferation and survival: cyclin dependent kinase 1 (CDK1), inhibitor of growth 4 (ING4), and early B-cell factor 3 (EBF3). This study underscores the importance of miR-650 in CLL biology and normal B-cell physiology.


Assuntos
Regulação da Expressão Gênica , Rearranjo Gênico , Genes de Imunoglobulinas , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Ciclo Celular/genética , Linhagem Celular Tumoral , Feminino , Genes de Cadeia Leve de Imunoglobulina , Humanos , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico
13.
Blood ; 119(19): 4467-75, 2012 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-22415752

RESUMO

Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.


Assuntos
Leucemia Linfocítica Crônica de Células B/classificação , Leucemia Linfocítica Crônica de Células B/genética , Técnicas de Diagnóstico Molecular/métodos , Terapia de Alvo Molecular , Receptores de Antígenos de Linfócitos B/genética , Sequência de Aminoácidos , Rearranjo Gênico do Linfócito B/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Receptores de Antígenos de Linfócitos B/metabolismo , Hipermutação Somática de Imunoglobulina/genética
14.
Haematologica ; 99(2): 329-38, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24038023

RESUMO

In chronic lymphocytic leukemia, usually a monoclonal disease, multiple productive immunoglobulin heavy chain gene rearrangements are identified sporadically. Prognostication of such cases based on immunoglobulin heavy variable gene mutational status can be problematic, especially if the different rearrangements have discordant mutational status. To gain insight into the possible biological mechanisms underlying the origin of the multiple rearrangements, we performed a comprehensive immunogenetic and immunophenotypic characterization of 31 cases with the multiple rearrangements identified in a cohort of 1147 patients with chronic lymphocytic leukemia. For the majority of cases (25/31), we provide evidence of the co-existence of at least two B lymphocyte clones with a chronic lymphocytic leukemia phenotype. We also identified clonal drifts in serial samples, likely driven by selection forces. More specifically, higher immunoglobulin variable gene identity to germline and longer complementarity determining region 3 were preferred in persistent or newly appearing clones, a phenomenon more pronounced in patients with stereotyped B-cell receptors. Finally, we report that other factors, such as TP53 gene defects and therapy administration, influence clonal selection. Our findings are relevant to clonal evolution in the context of antigen stimulation and transition of monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia.


Assuntos
Linfócitos B , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Leucemia Linfocítica Crônica de Células B , Mutação , Hipermutação Somática de Imunoglobulina , Proteína Supressora de Tumor p53/genética , Intervalo Livre de Doença , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Taxa de Sobrevida
15.
Geroscience ; 46(3): 3005-3019, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38172489

RESUMO

Biological age is typically estimated using biomarkers whose states have been observed to correlate with chronological age. A persistent limitation of such aging clocks is that it is difficult to establish how the biomarker states are related to the mechanisms of aging. Somatic mutations could potentially form the basis for a more fundamental aging clock since the mutations are both markers and drivers of aging and have a natural timescale. Cell lineage trees inferred from these mutations reflect the somatic evolutionary process, and thus, it has been conjectured, the aging status of the body. Such a timer has been impractical thus far, however, because detection of somatic variants in single cells presents a significant technological challenge. Here, we show that somatic mutations detected using single-cell RNA sequencing (scRNA-seq) from thousands of cells can be used to construct a cell lineage tree whose structure correlates with chronological age. De novo single-nucleotide variants (SNVs) are detected in human peripheral blood mononuclear cells using a modified protocol. A default model based on penalized multiple regression of chronological age on 31 metrics characterizing the phylogenetic tree gives a Pearson correlation of 0.81 and a median absolute error of ~4 years between predicted and chronological ages. Testing of the model on a public scRNA-seq dataset yields a Pearson correlation of 0.85. In addition, cell tree age predictions are found to be better predictors of certain clinical biomarkers than chronological age alone, for instance glucose, albumin levels, and leukocyte count. The geometry of the cell lineage tree records the structure of somatic evolution in the individual and represents a new modality of aging timer. In addition to providing a numerical estimate of "cell tree age," it unveils a temporal history of the aging process, revealing how clonal structure evolves over life span. Cell Tree Rings complements existing aging clocks and may help reduce the current uncertainty in the assessment of geroprotective trials.


Assuntos
Envelhecimento , Leucócitos Mononucleares , Humanos , Filogenia , Envelhecimento/genética , Longevidade , Biomarcadores
16.
Leukemia ; 38(7): 1455-1468, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38755420

RESUMO

In chronic lymphocytic leukemia (CLL), analysis of TP53 aberrations (deletion and/or mutation) is a crucial part of treatment decision-making algorithms. Technological and treatment advances have resulted in the need for an update of the last recommendations for TP53 analysis in CLL, published by ERIC, the European Research Initiative on CLL, in 2018. Based on the current knowledge of the relevance of low-burden TP53-mutated clones, a specific variant allele frequency (VAF) cut-off for reporting TP53 mutations is no longer recommended, but instead, the need for thorough method validation by the reporting laboratory is emphasized. The result of TP53 analyses should always be interpreted within the context of available laboratory and clinical information, treatment indication, and therapeutic options. Methodological aspects of introducing next-generation sequencing (NGS) in routine practice are discussed with a focus on reliable detection of low-burden clones. Furthermore, potential interpretation challenges are presented, and a simplified algorithm for the classification of TP53 variants in CLL is provided, representing a consensus based on previously published guidelines. Finally, the reporting requirements are highlighted, including a template for clinical reports of TP53 aberrations. These recommendations are intended to assist diagnosticians in the correct assessment of TP53 mutation status, but also physicians in the appropriate understanding of the lab reports, thus decreasing the risk of misinterpretation and incorrect management of patients in routine practice whilst also leading to improved stratification of patients with CLL in clinical trials.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Linfocítica Crônica de Células B , Mutação , Proteína Supressora de Tumor p53 , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/diagnóstico , Humanos , Proteína Supressora de Tumor p53/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/normas
17.
Mol Oncol ; 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38770541

RESUMO

Early identification of resistant cancer cells is currently a major challenge, as their expansion leads to refractoriness. To capture the dynamics of these cells, we made a comprehensive analysis of disease progression and treatment response in a chronic lymphocytic leukemia (CLL) patient using a combination of single-cell and bulk genomic methods. At diagnosis, the patient presented with unfavorable genetic markers, including notch receptor 1 (NOTCH1) mutation and loss(11q). The initial and subsequent treatment lines did not lead to a durable response and the patient developed refractory disease. Refractory CLL cells featured substantial dysregulation in B-cell phenotypic markers such as human leukocyte antigen (HLA) genes, immunoglobulin (IG) genes, CD19 molecule (CD19), membrane spanning 4-domains A1 (MS4A1; previously known as CD20), CD79a molecule (CD79A) and paired box 5 (PAX5), indicating B-cell de-differentiation and disease transformation. We described the clonal evolution and characterized in detail two cell populations that emerged during the refractory disease phase, differing in the presence of high genomic complexity. In addition, we successfully tracked the cells with high genomic complexity back to the time before treatment, where they formed a rare subpopulation. We have confirmed that single-cell RNA sequencing enables the characterization of refractory cells and the monitoring of their development over time.

18.
Ann Hematol ; 92(2): 249-54, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23014659

RESUMO

Allogeneic stem cell transplantation (SCT) is a treatment option for patients with poor-risk chronic lymphocytic leukemia (CLL). Sequential use of chemotherapy and reduced-intensity conditioning has been proposed to improve the treatment outcomes. Fludarabine (30 mg/m(2)/day) and cytarabine (2 g/m(2)/day) for 4 days (combination of fludarabine with cytarabine; FAraC) were used for cytoreduction. After 3 days of rest, reduced intensity conditioning (RIC) was carried out consisting of 4 Gy total body irradiation, 10-20 mg/kg/day antithymocyte globulin for 3 days, and 40-60 mg/kg/day cyclophosphamide for 2 days. The median time of neutrophil engraftment was 16 days. The most frequent toxicities were grades III/IV infections in 12 of 15 cases and gastrointestinal toxicities in 8 of 15 cases. Remission (complete remission + partial remission) was achieved in 14 of 15 patients (93 %), minimal residual disease negativity according to flowcytometric analysis was observed in 10 patients. Nonrelapse mortality after 1 and 2 years was 7 and 13 %, respectively. After the median follow-up from SCT of 30 months, 80 % of patients were alive (12/15), three patients have died, and three relapses occurred. The FAraC-RIC protocol seems to be a promising approach to the treatment of poor-risk CLL with a high response rate of 93 % and favorable progression-free survival and overall survival of 70 and 85 % at 2 years after SCT, respectively. Other prospective clinical trials are needed to confirm the results of this novel therapeutic strategy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Transplante de Células-Tronco de Sangue Periférico , Condicionamento Pré-Transplante/métodos , Adulto , Alemtuzumab , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Murinos/administração & dosagem , Transplante de Medula Óssea/efeitos adversos , Ciclofosfamida/administração & dosagem , Citarabina/administração & dosagem , Dexametasona/administração & dosagem , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Imunossupressores/uso terapêutico , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/cirurgia , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Prednisolona/administração & dosagem , Estudos Prospectivos , Recidiva , Risco , Rituximab , Terapia de Salvação , Condicionamento Pré-Transplante/efeitos adversos , Transplante Homólogo , Resultado do Tratamento , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados , Vincristina/administração & dosagem , Irradiação Corporal Total
19.
Biochim Biophys Acta Mol Cell Res ; 1870(7): 119489, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37271223

RESUMO

The cytokine interleukin-6 (IL-6) has considerable pro-inflammatory properties and is a driver of many physiological and pathophysiological processes. Cellular responses to IL-6 are mediated by membrane-bound or soluble forms of the IL-6 receptor (IL-6R) complexed with the signal-transducing subunit gp130. While expression of the membrane-bound IL-6R is restricted to selected cell types, soluble IL-6R (sIL-6R) enables gp130 engagement on all cells, a process termed IL-6 trans-signalling and considered to be pro-inflammatory. sIL-6R is predominantly generated through proteolytic processing by the metalloproteinase ADAM17. ADAM17 also liberates ligands of the epidermal growth factor receptor (EGFR), which is a prerequisite for EGFR activation and results in stimulation of proliferative signals. Hyperactivation of EGFR mostly due to activating mutations drives cancer development. Here, we reveal an important link between overshooting EGFR signalling and the IL-6 trans-signalling pathway. In epithelial cells, EGFR activity induces not only IL-6 expression but also the proteolytic release of sIL-6R from the cell membrane by increasing ADAM17 surface activity. We find that this derives from the transcriptional upregulation of iRhom2, a crucial regulator of ADAM17 trafficking and activation, upon EGFR engagement, which results in increased surface localization of ADAM17. Also, phosphorylation of the EGFR-downstream mediator ERK mediates ADAM17 activity via interaction with iRhom2. In sum, our study reveals an unforeseen interplay between EGFR activation and IL-6 trans-signalling, which has been shown to be fundamental in inflammation and cancer.


Assuntos
Proteína ADAM17 , Interleucina-6 , Transdução de Sinais , Receptor gp130 de Citocina/genética , Células Epiteliais/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Transdução de Sinais/genética , Humanos
20.
Cell Mol Gastroenterol Hepatol ; 15(6): 1391-1419, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36868311

RESUMO

BACKGROUND & AIMS: Patient-derived organoid cancer models are generated from epithelial tumor cells and reflect tumor characteristics. However, they lack the complexity of the tumor microenvironment, which is a key driver of tumorigenesis and therapy response. Here, we developed a colorectal cancer organoid model that incorporates matched epithelial cells and stromal fibroblasts. METHODS: Primary fibroblasts and tumor cells were isolated from colorectal cancer specimens. Fibroblasts were characterized for their proteome, secretome, and gene expression signatures. Fibroblast/organoid co-cultures were analyzed by immunohistochemistry and compared with their tissue of origin, as well as on gene expression levels compared with standard organoid models. Bioinformatics deconvolution was used to calculate cellular proportions of cell subsets in organoids based on single-cell RNA sequencing data. RESULTS: Normal primary fibroblasts, isolated from tumor adjacent tissue, and cancer associated fibroblasts retained their molecular characteristics in vitro, including higher motility of cancer associated compared with normal fibroblasts. Importantly, both cancer-associated fibroblasts and normal fibroblasts supported cancer cell proliferation in 3D co-cultures, without the addition of classical niche factors. Organoids grown together with fibroblasts displayed a larger cellular heterogeneity of tumor cells compared with mono-cultures and closely resembled the in vivo tumor morphology. Additionally, we observed a mutual crosstalk between tumor cells and fibroblasts in the co-cultures. This was manifested by considerably deregulated pathways such as cell-cell communication and extracellular matrix remodeling in the organoids. Thrombospondin-1 was identified as a critical factor for fibroblast invasiveness. CONCLUSION: We developed a physiological tumor/stroma model, which will be vital as a personalized tumor model to study disease mechanisms and therapy response in colorectal cancer.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias Colorretais , Humanos , Fibroblastos/metabolismo , Técnicas de Cocultura , Organoides/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Neoplasias Colorretais/patologia , Microambiente Tumoral
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