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1.
Clin Cancer Res ; 30(12): 2659-2671, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38619278

RESUMO

PURPOSE: The inherent genetic heterogeneity of acute myeloid leukemia (AML) has challenged the development of precise and effective therapies. The objective of this study was to elucidate the genomic basis of drug resistance or sensitivity, identify signatures for drug response prediction, and provide resources to the research community. EXPERIMENTAL DESIGN: We performed targeted sequencing, high-throughput drug screening, and single-cell genomic profiling on leukemia cell samples derived from patients with AML. Statistical approaches and machine learning models were applied to identify signatures for drug response prediction. We also integrated large public datasets to understand the co-occurring mutation patterns and further investigated the mutation profiles in the single cells. The features revealed in the co-occurring or mutual exclusivity pattern were further subjected to machine learning models. RESULTS: We detected genetic signatures associated with sensitivity or resistance to specific agents, and identified five co-occurring mutation groups. The application of single-cell genomic sequencing unveiled the co-occurrence of variants at the individual cell level, highlighting the presence of distinct subclones within patients with AML. Using the mutation pattern for drug response prediction demonstrates high accuracy in predicting sensitivity to some drug classes, such as MEK inhibitors for RAS-mutated leukemia. CONCLUSIONS: Our study highlights the importance of considering the gene mutation patterns for the prediction of drug response in AML. It provides a framework for categorizing patients with AML by mutations that enable drug sensitivity prediction.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda , Mutação , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Análise de Célula Única/métodos , Aprendizado de Máquina , Sequenciamento de Nucleotídeos em Larga Escala , Masculino
2.
Cell Genom ; 4(2): 100487, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38278156

RESUMO

Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or nuclear factor κB (NF-κB) inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.


Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Transdução de Sinais , Receptores Proteína Tirosina Quinases/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Genômica , Proteínas Serina-Treonina Quinases , Proteínas de Ciclo Celular/uso terapêutico
3.
bioRxiv ; 2023 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-37398090

RESUMO

Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or NF-kB inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.

4.
Int J Cancer ; 153(1): 183-196, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-36912284

RESUMO

Fanconi anemia (FA) is a heritable malformation, bone marrow failure and cancer predisposition syndrome that confers an exceptionally high risk of squamous carcinomas. These carcinomas originate in epithelia lining the mouth, proximal esophagus, vulva and anus: their origins are not understood, and no effective ways have been identified to prevent or delay their appearance. Many FA-associated carcinomas are also therapeutically challenging: they may be multi-focal and stage-advanced at diagnosis, and most individuals with FA cannot tolerate standard-of-care systemic therapies such as DNA cross-linking drugs or ionizing radiation due to constitutional DNA damage hypersensitivity. We developed the Fanconi Anemia Cancer Cell Line Resource (FA-CCLR) to foster new work on the origins, treatment and prevention of FA-associated carcinomas. The FA-CCLR consists of Fanconi-isogenic head and neck squamous cell carcinoma (HNSCC) cell line pairs generated from five individuals with FA-associated HNSCC, and five individuals with sporadic HNSCC. Sporadic, isogenic HNSCC cell line pairs were generated in parallel with FA patient-derived isogenic cell line pairs to provide comparable experimental material to use to identify cell and molecular phenotypes driven by germline or somatic loss of Fanconi pathway function, and the subset of these FA-dependent phenotypes that can be modified, complemented or suppressed. All 10 FANC-isogenic cell line pairs are available to academic, non-profit and industry investigators via the "Fanconi Anemia Research Materials" Resource and Repository at Oregon Health & Sciences University, Portland OR.


Assuntos
Carcinoma de Células Escamosas , Anemia de Fanconi , Neoplasias de Cabeça e Pescoço , Feminino , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Anemia de Fanconi/genética , Anemia de Fanconi/complicações , Anemia de Fanconi/patologia , Ciência Translacional Biomédica , Neoplasias de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral
6.
Cell Rep ; 38(3): 110269, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-35045296

RESUMO

Cells are complex systems in which many functions are performed by different genetically defined and encoded functional modules. To systematically understand how these modules respond to drug or genetic perturbations, we develop a functional module states framework. Using this framework, we (1) define the drug-induced transcriptional state space for breast cancer cell lines using large public gene expression datasets and reveal that the transcriptional states are associated with drug concentration and drug targets, (2) identify potential targetable vulnerabilities through integrative analysis of transcriptional states after drug treatment and gene knockdown-associated cancer dependency, and (3) use functional module states to predict transcriptional state-dependent drug sensitivity and build prediction models for drug response. This approach demonstrates a similar prediction performance as approaches using high-dimensional gene expression values, with the added advantage of more clearly revealing biologically relevant transcriptional states and key regulators.


Assuntos
Neoplasias da Mama , Perfilação da Expressão Gênica/métodos , Aprendizado de Máquina , Terapia de Alvo Molecular , Transcriptoma , Feminino , Humanos
7.
PLoS Comput Biol ; 17(12): e1009626, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34968384

RESUMO

Identification of cell phenotypic states within heterogeneous populations, along with elucidation of their switching dynamics, is a central challenge in modern biology. Conventional single-cell analysis methods typically provide only indirect, static phenotypic readouts. Transmitted light images, on the other hand, provide direct morphological readouts and can be acquired over time to provide a rich data source for dynamic cell phenotypic state identification. Here, we describe an end-to-end deep learning platform, UPSIDE (Unsupervised Phenotypic State IDEntification), for discovering cell states and their dynamics from transmitted light movies. UPSIDE uses the variational auto-encoder architecture to learn latent cell representations, which are then clustered for state identification, decoded for feature interpretation, and linked across movie frames for transition rate inference. Using UPSIDE, we identified distinct blood cell types in a heterogeneous dataset. We then analyzed movies of patient-derived acute myeloid leukemia cells, from which we identified stem-cell associated morphological states as well as the transition rates to and from these states. UPSIDE opens up the use of transmitted light movies for systematic exploration of cell state heterogeneity and dynamics in biology and medicine.


Assuntos
Células Sanguíneas/classificação , Células Sanguíneas/citologia , Microscopia/métodos , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/métodos , Aprendizado de Máquina não Supervisionado , Algoritmos , Células Sanguíneas/patologia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Leucemia Mieloide Aguda/patologia , Luz , Fenótipo
8.
Mol Syst Biol ; 16(6): e9442, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32500953

RESUMO

Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates hundreds of thousands of live cells based on their visual phenotype. Automated imaging and phenotypic analysis directs selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence-activated cell sorting. First, we use Visual Cell Sorting to assess hundreds of nuclear localization sequence variants in a pooled format, identifying variants that improve nuclear localization and enabling annotation of nuclear localization sequences in thousands of human proteins. Second, we recover cells that retain normal nuclear morphologies after paclitaxel treatment, and then derive their single-cell transcriptomes to identify pathways associated with paclitaxel resistance in cancers. Unlike alternative methods, Visual Cell Sorting depends on inexpensive reagents and commercially available hardware. As such, it can be readily deployed to uncover the relationships between visual cellular phenotypes and internal states, including genotypes and gene expression programs.


Assuntos
Células/citologia , Microscopia de Fluorescência/instrumentação , Linhagem Celular , Forma do Núcleo Celular/efeitos dos fármacos , Citometria de Fluxo , Testes Genéticos , Humanos , Sinais de Localização Nuclear/metabolismo , Paclitaxel/farmacologia , Fenótipo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
9.
Nat Rev Cancer ; 20(9): 533-549, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32472073

RESUMO

Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.


Assuntos
Interação Gene-Ambiente , Predisposição Genética para Doença , Neoplasias/genética , Animais , Mutação em Linhagem Germinativa , Humanos
10.
Hum Gene Ther ; 30(7): 814-828, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30793977

RESUMO

This study identified 35 new sites for targeted transgene insertion that have the potential to serve as new human genomic "safe harbor" sites (SHS). SHS potential for these 35 sites, located on 16 chromosomes, including both arms of the human X chromosome, and for the existing human SHS AAVS1, hROSA26, and CCR5 was assessed using eight different desirable, widely accepted criteria for SHS verifiable with human genomic data. Three representative newly identified sites on human chromosomes 2 and 4 were then experimentally validated by in vitro and in vivo cleavage-sensitivity tests, and analyzed for population-level and cell line-specific sequence variants that might confound site targeting. The highly ranked site on chromosome 4 (SHS231) was further characterized by targeted homology-dependent and -independent transgene insertion and expression in different human cell lines. The structure and fidelity of transgene insertions at this site were confirmed, together with analyses that demonstrated stable expression and function of transgene-encoded proteins, including fluorescent protein markers, selectable marker cassettes, and Cas9 protein variants. SHS-integrated transgene-encoded Cas9 proteins were shown to be capable of introducing a large (17 kb) gRNA-specified deletion in the PAX3/FOXO1 fusion oncogene in human rhabdomyosarcoma cells and as a Cas9-VPR fusion protein to upregulate expression of the muscle-specific transcription factor MYF5 in human rhabdomyosarcoma cells. An engineering "toolkit" was developed to enable easy use of the most extensively characterized of these new human sites, SHS231, located on the proximal long arm of chromosome 4. The target sites identified here have the potential to serve as additional human SHS to enable basic and clinical gene editing and genome-engineering applications.


Assuntos
Cromossomos Humanos , Mutagênese Insercional , Transgenes , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular , Pontos de Quebra do Cromossomo , Edição de Genes , Expressão Gênica , Técnicas de Introdução de Genes , Marcação de Genes , Loci Gênicos , Genoma Humano , Mapeamento Geográfico , Humanos
11.
DNA Repair (Amst) ; 72: 1-9, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30389308

RESUMO

Formaldehyde is a ubiquitous DNA damaging agent, with human exposures occurring from both exogenous and endogenous sources. Formaldehyde exposure can result in multiple types of DNA damage, including DNA-protein crosslinks and thus, is representative of other exposures that induce DNA-protein crosslinks such as cigarette smoke, automobile exhaust, wood smoke, metals, ionizing radiation, and certain chemotherapeutics. Our objective in this study was to identify the genes necessary to mitigate formaldehyde toxicity following chronic exposure in human cells. We used siRNAs that targeted 320 genes representing all major human DNA repair and damage response pathways, in order to assess cell proliferation following siRNA depletion and subsequent formaldehyde treatment. Three unrelated human cell lines frequently used in genotoxicity studies (SW480, U-2 OS and GM00639) were used to identify common pathways involved in mitigating formaldehyde sensitivity. Although there were gene-specific differences among the cell lines, four inter-related cellular pathways were determined to mitigate formaldehyde toxicity: homologous recombination, DNA double-strand break repair, ionizing radiation response and DNA replication. Additional insight into cell line-specific response patterns was obtained by using a combination of exome sequencing and Cancer Cell Line Encyclopedia genomic data. The results of this DNA damage repair pathway-focused siRNA screen for formaldehyde toxicity in human cells provide a foundation for detailed mechanistic analyses of pathway-specific involvement in the response to environmentally-induced DNA-protein crosslinks and, more broadly, genotoxicity studies using human and other mammalian cell lines.


Assuntos
Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Formaldeído/toxicidade , Interferência de RNA , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Genômica , Humanos
12.
Cell Rep ; 23(1): 239-254.e6, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29617664

RESUMO

DNA damage repair (DDR) pathways modulate cancer risk, progression, and therapeutic response. We systematically analyzed somatic alterations to provide a comprehensive view of DDR deficiency across 33 cancer types. Mutations with accompanying loss of heterozygosity were observed in over 1/3 of DDR genes, including TP53 and BRCA1/2. Other prevalent alterations included epigenetic silencing of the direct repair genes EXO5, MGMT, and ALKBH3 in ∼20% of samples. Homologous recombination deficiency (HRD) was present at varying frequency in many cancer types, most notably ovarian cancer. However, in contrast to ovarian cancer, HRD was associated with worse outcomes in several other cancers. Protein structure-based analyses allowed us to predict functional consequences of rare, recurrent DDR mutations. A new machine-learning-based classifier developed from gene expression data allowed us to identify alterations that phenocopy deleterious TP53 mutations. These frequent DDR gene alterations in many human cancers have functional consequences that may determine cancer progression and guide therapy.


Assuntos
Genoma Humano , Neoplasias/genética , Reparo de DNA por Recombinação , Linhagem Celular Tumoral , Dano ao DNA , Inativação Gênica , Humanos , Perda de Heterozigosidade , Aprendizado de Máquina , Mutação , Neoplasias/classificação , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
13.
Ageing Res Rev ; 41: 82-97, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29146545

RESUMO

Werner syndrome (WS) is a heritable autosomal recessive human disorder characterized by the premature onset of several age-associated pathologies including cancer. The protein defective in WS patients, WRN, is encoded by a member of the human RECQ gene family that contains both a DNA exonuclease and a helicase domain. WRN has been shown to participate in several DNA metabolic pathways including DNA replication, recombination and repair, as well as telomere maintenance and transcription modulation. Here we review base pair-level genetic variation that has been documented in WRN, with an emphasis on non-synonymous coding single nucleotide polymorphisms (SNPs) and their associations with anthropomorphic features, longevity and disease risk. These associations have been challenging to identify, as many reported WRN SNP associations appear to be further conditioned upon ethnic, age, gender or other environmental co-variables. The WRN variant phenotypic associations identified to date are intriguing, and several are of clear clinical import. Consequently, it will be important to extend these initial associations and to identify the mechanisms and conditions under which specific WRN variants may compromise WRN function to drive cellular and organismal phenotypes as well as disease risk.


Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , Estudos de Associação Genética , Variação Genética/fisiologia , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Envelhecimento/patologia , Animais , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Estudos de Associação Genética/tendências , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Síndrome de Werner/diagnóstico , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo
14.
Sci Rep ; 7: 44081, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28276523

RESUMO

Loss-of-function mutations in the WRN helicase gene cause Werner syndrome- a progeroid syndrome with an elevated risk of cancer and other age-associated diseases. Large numbers of single nucleotide polymorphisms have been identified in WRN. We report here the organismal, cellular, and molecular phenotypes of variant rs3087425 (c. 2500C > T) that results in an arginine to cysteine substitution at residue 834 (R834C) and up to 90% reduction of WRN helicase activity. This variant is present at a high (5%) frequency in Mexico, where we identified 153 heterozygous and three homozygous individuals among 3,130 genotyped subjects. Family studies of probands identified ten additional TT homozygotes. Biochemical analysis of WRN protein purified from TT lymphoblast cell lines confirmed that the R834C substitution strongly and selectively reduces WRN helicase, but not exonuclease activity. Replication track analyses showed reduced replication fork progression in some homozygous cells following DNA replication stress. Among the thirteen TT homozygotes, we identified a previously unreported and statistically significant gender bias in favor of males (p = 0.0016), but none of the clinical findings associated with Werner syndrome. Our results indicate that WRN helicase activity alone is not rate-limiting for the development of clinical WS.


Assuntos
Homozigoto , Mutação de Sentido Incorreto , Fenótipo , Helicase da Síndrome de Werner/metabolismo , Síndrome de Werner/genética , Adolescente , Adulto , Idoso , Substituição de Aminoácidos , Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de Werner/enzimologia , Síndrome de Werner/patologia , Helicase da Síndrome de Werner/genética
15.
Ageing Res Rev ; 33: 105-114, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26993153

RESUMO

Werner syndrome (WS) is a prototypical segmental progeroid syndrome characterized by multiple features consistent with accelerated aging. It is caused by null mutations of the WRN gene, which encodes a member of the RECQ family of DNA helicases. A unique feature of the WRN helicase is the presence of an exonuclease domain in its N-terminal region. Biochemical and cell biological studies during the past decade have demonstrated involvements of the WRN protein in multiple DNA transactions, including DNA repair, recombination, replication and transcription. A role of the WRN protein in telomere maintenance could explain many of the WS phenotypes. Recent discoveries of new progeroid loci found in atypical Werner cases continue to support the concept of genomic instability as a major mechanism of biological aging. Based on these biological insights, efforts are underway to develop therapeutic interventions for WS and related progeroid syndromes.


Assuntos
Senilidade Prematura , Helicase da Síndrome de Werner/genética , Síndrome de Werner , Senilidade Prematura/genética , Senilidade Prematura/metabolismo , Reparo do DNA , Replicação do DNA , Exodesoxirribonucleases , Humanos , Mutação , Síndrome de Werner/diagnóstico , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Síndrome de Werner/fisiopatologia
16.
Hum Mutat ; 38(2): 193-203, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27859906

RESUMO

Heritable loss of function mutations in the human RECQ helicase genes BLM, WRN, and RECQL4 cause Bloom, Werner, and Rothmund-Thomson syndromes, cancer predispositions with additional developmental or progeroid features. In order to better understand RECQ pathogenic and population variation, we systematically analyzed genetic variation in all five human RECQ helicase genes. A total of 3,741 unique base pair-level variants were identified, across 17,605 potential mutation sites. Direct counting of BLM, RECQL4, and WRN pathogenic variants was used to determine aggregate and disease-specific carrier frequencies. The use of biochemical and model organism data, together with computational prediction, identified over 300 potentially pathogenic population variants in RECQL and RECQL5, the two RECQ helicases that are not yet linked to a heritable deficiency syndrome. Despite the presence of these predicted pathogenic variants in the human population, we identified no individuals homozygous for any biochemically verified or predicted pathogenic RECQL or RECQL5 variant. Nor did we find any individual heterozygous for known pathogenic variants in two or more of the disease-associated RECQ helicase genes BLM, RECQL4, or WRN. Several postulated RECQ helicase deficiency syndromes-RECQL or RECQL5 loss of function, or compound haploinsufficiency for the disease-associated RECQ helicases-may remain missing, as they likely incompatible with life.


Assuntos
Estudos de Associação Genética , Genética Populacional , Mutação , RecQ Helicases/genética , Biologia Computacional/métodos , Análise Mutacional de DNA , Bases de Dados de Ácidos Nucleicos , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Curva ROC , Software , Navegador
17.
Blood ; 128(24): 2774-2784, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27756748

RESUMO

Fanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2-/- mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2-/- mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2-/-Trp53+/- mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient-derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells.


Assuntos
Carcinogênese/patologia , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/patologia , Hematopoese/efeitos dos fármacos , Metformina/farmacologia , Aldeídos/metabolismo , Animais , Contagem de Células Sanguíneas , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Carcinogênese/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Quebra Cromossômica , Dano ao DNA , Dieta , Anemia de Fanconi/sangue , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Guanidinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Inativação Metabólica/efeitos dos fármacos , Metformina/administração & dosagem , Camundongos , Poli I-C/farmacologia
18.
Sci Rep ; 6: 32038, 2016 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-27559010

RESUMO

Werner syndrome (WS) is the canonical adult human progeroid ('premature aging') syndrome. Patients with this autosomal recessive Mendelian disorder display constitutional genomic instability and an elevated risk of important age-associated diseases including cancer. Remarkably few analyses of WS patient tissue and tumors have been performed to provide insight into WS disease pathogenesis or the high risk of neoplasia. We used autopsy tissue from four mutation-typed WS patients to characterize pathologic and genomic features of WS, and to determine genomic features of three neoplasms arising in two of these patients. The results of these analyses provide new information on WS pathology and genomics; provide a first genomic characterization of neoplasms arising in WS; and provide new histopathologic and genomic data to test several popular models of WS disease pathogenesis.


Assuntos
Mutação , Síndrome de Werner/genética , Síndrome de Werner/patologia , Adulto , Senescência Celular , DNA Mitocondrial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Telômero/genética , Helicase da Síndrome de Werner/genética
19.
Clin Cancer Res ; 22(18): 4612-22, 2016 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-27121793

RESUMO

PURPOSE: WRN promoter CpG island hypermethylation in colorectal cancer has been reported to increase sensitivity to irinotecan-based therapies. We aimed to characterize methylation of the WRN promoter, determine the effect of WRN promoter hypermethylation upon expression, and validate a previous report that WRN promoter hypermethylation predicts improved outcomes for patients with metastatic colorectal cancer (mCRC) treated with irinotecan-based therapy. EXPERIMENTAL DESIGN: WRN methylation status was assessed using methylation-specific PCR and bisulfite sequencing assays. WRN expression was determined using qRT-PCR and Western blotting. WRN methylation status was correlated with overall survival (OS) and progression-free survival (PFS) in 183 patients with mCRC. Among these patients, 90 received capecitabine monotherapy as first-line therapy, and 93 received capecitabine plus irinotecan (CAPIRI) therapy as part of the CAIRO phase III clinical trial. RESULTS: WRN mRNA and WRN protein expression levels were low in colorectal cancer cell lines and in primary colorectal cancer and were largely independent of WRN methylation status. Patients with methylated WRN colorectal cancer had a shorter OS compared with patients who had unmethylated WRN colorectal cancer (HR = 1.6; 95% confidence interval [CI], 1.2-2.2; P = 0.003). Patients with unmethylated WRN showed a significantly longer PFS when treated with CAPIRI compared with capecitabine alone (HR = 0.48; 95% CI, 0.32-0.70; P = 0.0001). In contrast, patients did not benefit from adding irinotecan to capecitabine when WRN was methylated (HR = 1.1; 95% CI, 0.69-1.77; P = 0.7). CONCLUSIONS: WRN expression is largely independent of WRN promoter hypermethylation in colorectal cancer. Moreover, we could not validate the previous finding that WRN promoter hypermethylation predicts improved clinical outcomes of mCRC treated with irinotecan-based therapy and found instead the opposite result. Clin Cancer Res; 22(18); 4612-22. ©2016 AACR.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Ilhas de CpG , Metilação de DNA , Regiões Promotoras Genéticas , Helicase da Síndrome de Werner/genética , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Expressão Gênica , Humanos , Irinotecano , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais
20.
Hum Mol Genet ; 25(10): 2060-2069, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26984941

RESUMO

The Werner syndrome (WS) is a prototypic adult Mendelian progeroid syndrome in which signs of premature aging are associated with genomic instability and an elevated risk of cancer. The WRN RECQ helicase protein binds and unwinds G-quadruplex (G4) DNA substrates in vitro, and we identified significant enrichment in G4 sequence motifs at the transcription start site and 5' ends of first introns (false discovery rate < 0.001) of genes down-regulated in WS patient fibroblasts. This finding provides strong evidence that WRN binds G4 DNA structures at many chromosomal sites to modulate gene expression. WRN appears to bind a distinct subpopulation of G4 motifs in human cells, when compared with the related Bloom syndrome RECQ helicase protein. Functional annotation of the genes and miRNAs altered in WS provided new insight into WS disease pathogenesis. WS patient fibroblasts displayed altered expression of multiple, mechanistically distinct, senescence-associated gene expression programs, with altered expression of disease-associated miRNAs, and dysregulation of canonical pathways that regulate cell signaling, genome stability and tumorigenesis. WS fibroblasts also displayed a highly statistically significant and distinct gene expression signature, with coordinate overexpression of nearly all of the cytoplasmic tRNA synthetases and associated ARS-interacting multifunctional protein genes. The 'non-canonical' functions of many of these upregulated tRNA charging proteins may together promote WS disease pathogenesis. Our results identify the human WRN RECQ protein as a G4 helicase that modulates gene expression in G4-dependent fashion at many chromosomal sites and provide several new and unexpected mechanistic insights into WS disease pathogenesis.


Assuntos
Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Neoplasias/genética , RecQ Helicases/genética , Síndrome de Werner/genética , Carcinogênese/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos , Quadruplex G , Regulação da Expressão Gênica , Genoma Humano , Humanos , MicroRNAs , Neoplasias/patologia , Motivos de Nucleotídeos , RecQ Helicases/metabolismo
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