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AIMS: Somatic mutations of the epigenetic regulators DNMT3A and TET2 causing clonal expansion of haematopoietic cells (clonal haematopoiesis; CH) were shown to be associated with poor prognosis in chronic ischaemic heart failure (CHF). The aim of our analysis was to define a threshold of variant allele frequency (VAF) for the prognostic significance of CH in CHF. METHODS AND RESULTS: We analysed bone marrow and peripheral blood-derived cells from 419 patients with CHF by error-corrected amplicon sequencing. Cut-off VAFs were optimized by maximizing sensitivity plus specificity from a time-dependent receiver operating characteristic (ROC) curve analysis from censored data. 56.2% of patients were carriers of a DNMT3A- (N = 173) or a TET2- (N = 113) mutation with a VAF >0.5%, with 59 patients harbouring mutations in both genes. Survival ROC analyses revealed an optimized cut-off value of 0.73% for TET2- and 1.15% for DNMT3A-CH-driver mutations. Five-year-mortality was 18% in patients without any detected DNMT3A- or TET2 mutation (VAF < 0.5%), 29% with only one DNMT3A- or TET2-CH-driver mutations above the respective cut-off level and 42% in patients harbouring both DNMT3A- and TET2-CH-driver mutations above the respective cut-off levels. In carriers of a DNMT3A mutation with VAF ≥ 1.15%, 5-year mortality was 31%, compared with 18% mortality in those with VAF < 1.15% (P = 0.048). Likewise, in patients with TET2 mutations, 5-year mortality was 32% with VAF ≥ 0.73%, compared with 19% mortality with VAF < 0.73% (P = 0.029). CONCLUSION: The present study defines novel threshold levels for clone size caused by acquired somatic mutations in the CH-driver genes DNMT3A and TET2 that are associated with worse outcome in patients with CHF.
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Hematopoiese Clonal , Insuficiência Cardíaca , Células Clonais , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Dioxigenases , Humanos , Mutação , Prognóstico , Proteínas Proto-Oncogênicas/genéticaRESUMO
This study focused on the effects induced by a random positioning machine (RPM) on FTC-133 thyroid cancer cells and evaluated signaling elements involved in 3-dimensional multicellular tumor spheroid (MCTS) formation. The cells were cultured on the RPM, a device developed to simulate microgravity, and under static 1-g conditions. After 24 h on the RPM, MCTSs swimming in culture supernatants were found, in addition to growth of adherent (AD) cells. Cells grown on the RPM showed higher levels of NF-κB p65 protein and apoptosis than 1-g controls, a result also found earlier in endothelial cells. Employing microarray analysis, we found 487 significantly regulated transcripts belonging not only to the apoptosis pathway but also to other biological processes. Selected transcripts were analyzed with quantitative real-time PCR using the same samples. Compared with 1-g IL-6, IL-8, CD44, and OPN were significantly up-regulated in AD cells but not in MCTSs, while ERK1/2, CAV2, TLN1, and CTGF were significantly down-regulated in AD cells. Simultaneously, the expression of ERK2, IL-6, CAV2, TLN1, and CTGF was reduced in MCTSs. IL-6 protein expression and secretion mirrored its gene expression. Thus, we concluded that the signaling elements IL-6, IL-8, OPN, TLN1, and CTGF are involved with NF-κB p65 in RPM-dependent thyroid carcinoma cell spheroid formation.
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Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Gravitação , Esferoides Celulares/metabolismo , Neoplasias da Glândula Tireoide/genética , Apoptose/genética , Western Blotting , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Análise por Conglomerados , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Osteopontina/genética , Osteopontina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Esferoides Celulares/patologia , Talina/genética , Talina/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4Gy in both RET/PTC-positive systems but no common genes at 62.5mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent.
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Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Processamento Pós-Transcricional do RNA , Glândula Tireoide/efeitos da radiação , Neoplasias da Glândula Tireoide/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Raios XRESUMO
Autophagy is an important survival mechanism that allows recycling of nutrients and removal of damaged organelles and has been shown to contribute to the proliferation of acute myeloid leukemia (AML) cells. However, little is known about the mechanism by which autophagy- dependent AML cells can overcome dysfunctional autophagy. In our study we identified autophagy related protein 3 (ATG3) as a crucial autophagy gene for AML cell proliferation by conducting a CRISPR/Cas9 dropout screen with a library targeting around 200 autophagy-related genes. shRNA-mediated loss of ATG3 impaired autophagy function in AML cells and increased their mitochondrial activity and energy metabolism, as shown by elevated mitochondrial ROS generation and mitochondrial respiration. Using tracer-based NMR metabolomics analysis we further demonstrate that the loss of ATG3 resulted in an upregulation of glycolysis, lactate production, and oxidative phosphorylation. Additionally, loss of ATG3 strongly sensitized AML cells to the inhibition of mitochondrial metabolism. These findings highlight the metabolic vulnerabilities that AML cells acquire from autophagy inhibition and support further exploration of combination therapies targeting autophagy and mitochondrial metabolism in AML.
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AIMS: Somatic mutations in haematopoietic stem cells can lead to the clonal expansion of mutated blood cells, known as clonal haematopoiesis (CH). Mutations in the most prevalent driver genes DNMT3A and TET2 with a variant allele frequency (VAF) ≥ 2% have been associated with atherosclerosis and chronic heart failure of ischemic origin (CHF). However, the effects of mutations in other driver genes for CH with low VAF (<2%) on CHF are still unknown. METHODS AND RESULTS: Therefore, we analysed mononuclear bone marrow and blood cells from 399 CHF patients by deep error-corrected targeted sequencing of 56 genes and associated mutations with the long-term mortality in these patients (3.95 years median follow-up). We detected 1113 mutations with a VAF ≥ 0.5% in 347 of 399 patients, and only 13% had no detectable CH. Despite a high prevalence of mutations in the most frequently mutated genes DNMT3A (165 patients) and TET2 (107 patients), mutations in CBL, CEBPA, EZH2, GNB1, PHF6, SMC1A, and SRSF2 were associated with increased death compared with the average death rate of all patients. To avoid confounding effects, we excluded patients with DNMT3A-related, TET2-related, and other clonal haematopoiesis of indeterminate potential (CHIP)-related mutations with a VAF ≥ 2% for further analyses. Kaplan-Meier survival analyses revealed a significantly higher mortality in patients with mutations in either of the seven genes (53 patients), combined as the CH-risk gene set for CHF. Baseline patient characteristics showed no significant differences in any parameter including patient age, confounding diseases, severity of CHF, or blood cell parameters except for a reduced number of platelets in patients with mutations in the risk gene set in comparison with patients without. However, carrying a mutation in any of the risk genes remained significant after multivariate cox regression analysis (hazard ratio, 3.1; 95% confidence interval, 1.8-5.4; P < 0.001), whereas platelet numbers did not. CONCLUSIONS: Somatic mutations with low VAF in a distinct set of genes, namely, in CBL, CEBPA, EZH2, GNB1, PHF6, SMC1A, and SRSF2, are significantly associated with mortality in CHF, independently of the most prevalent CHIP-mutations in DNMT3A and TET2. Mutations in these genes are prevalent in young CHF patients and comprise an independent risk factor for the outcome of CHF, potentially providing a novel tool for risk assessment in CHF.
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Hematopoiese Clonal , Insuficiência Cardíaca , Proteínas de Ligação a DNA/genética , Insuficiência Cardíaca/genética , Humanos , Mutação , Proteínas Proto-Oncogênicas/genéticaRESUMO
Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein-protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.
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Carcinogênese/genética , Neoplasias do Colo/genética , Proteína 2 Glutamina gama-Glutamiltransferase/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Colo/patologia , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mapas de Interação de Proteínas/genética , Transcriptoma/genéticaRESUMO
Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneity between patients. To facilitate high-throughput genetic testing and functional identification of tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in human colon organoids. Using transforming growth factor ß (TGF-ß) resistance as a paradigm to establish sensitivity and scalability in vitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screening in 3D organoids. We then screened a pan-cancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC-/-;KRASG12D mutations, which were xenografted to study clonal advantages in context of a complex tumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediators of CRC growth. gRNAs were validated in a secondary screen using unique molecular identifiers (UMIs) to adjust for clonal drift and to distinguish clone size and abundance. Together, these findings highlight a powerful organoid-based platform for pooled CRISPR-Cas9 screening for patient-specific functional genomics.
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Sistemas CRISPR-Cas , Organoides , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Colo , Genes Supressores de Tumor , HumanosRESUMO
Several lines of evidence have linked limb teratogenesis to radiation-induced apoptosis and to the p53 status in murine fetuses. In previous reports, we studied the occurrence of various malformations after intrauterine irradiation and showed that these malformations were modulated by p53-deficiency as well as by the developmental stage at which embryos were irradiated. In this new study, we focused onto one particular phenotype namely forelimb defects to further unravel the cellular and molecular mechanisms underlying this malformation. We measured various parameters expected to be directly or indirectly influenced by irradiation damage. The mouse fetuses were irradiated at day 12 p.c. (post conception) and examined for forelimb defects on gestational days 15, 16, 17 and 19 of development. The release of inflammatory cytokines was determined in the amniotic fluid on day 16 p.c. and the mean telomere lengths assessed at days 12, 13 and 19 p.c. Differential gene expression within the forelimb bud tissues was determined using Real Time quantitative PCR (RTqPCR) 24 h following irradiation. Apoptosis was investigated in the normal and malformed fetuses using the TUNEL assay and RTqPCR. First, we found that irradiated fetuses with forelimb defects displayed excessive apoptosis in the predigital regions. Besides, overexpression of the pro-apoptotic Bax gene indicates a mitochondrial-mediated cell death. Secondly, our results showed overexpression of MKK3 and MKK7 (members of the stress-activated MAP kinase family) within the malformed fetuses. The latter could be involved in radiation-induced apoptosis through activation of the p38 and JNK pathways. Thirdly, we found that irradiated fetuses exhibiting forelimb defects showed a marked telomere shortening. Interestingly, telomere shortening was observed as the malformations became apparent. Fourthly, we measured cytokine levels in the amniotic fluid and detected a considerable inflammatory reaction among the irradiated fetuses as evidenced by the increase in pro-inflammatory cytokine levels. Altogether, our data suggest that transcriptional modulations of apoptotic, inflammation, stress, and DNA damage players are early events in radiation-induced forelimb defects. These changes resulted in harsh developmental conditions as indicated by a marked increase in cytokine levels in the amniotic fluid and telomere shortening, two features concomitant with the onset of the forelimb defect phenotype in our study.
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Anormalidades Induzidas por Radiação/metabolismo , Citocinas/metabolismo , Feto/metabolismo , Membro Anterior/metabolismo , Exposição Materna/efeitos adversos , Telômero/ultraestrutura , Anormalidades Induzidas por Radiação/etiologia , Anormalidades Induzidas por Radiação/patologia , Líquido Amniótico/metabolismo , Animais , Apoptose , Dano ao DNA , Feminino , Feto/anormalidades , Membro Anterior/anormalidades , Botões de Extremidades/citologia , Botões de Extremidades/efeitos da radiação , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Telômero/metabolismo , Proteína Supressora de Tumor p53RESUMO
We previously reported the establishment of a rare xenograft derived from a recurrent oligodendroglioma with 1p/19q codeletion. Here, we analyzed in detail the exome sequencing datasets from the recurrent oligodendroglioma (WHO grade III, recurrent O2010) and the first-generation xenograft (xenograft1). Somatic SNVs and small InDels (n = 80) with potential effects at the protein level in recurrent O2010 included variants in IDH1 (NM_005896:c.395G>A; p. Arg132His), FUBP1 (NM_003902:c.1307_1310delTAGA; p.Ile436fs), and CIC (NM_015125:c.4421T>G; p.Val1474Gly). All but 2 of these 80 variants were also present in xenograft1, along with 7 new variants. Deep sequencing of the 87 SNVs and InDels in the original tumor (WHO grade III, primary O2005) and in a second-generation xenograft (xenograft2) revealed that only 11 variants, including IDH1 (NM_005896:c.395G>A; p. Arg132His), PSKH1 (NM_006742.2:c.650G>A; p.Arg217Gln), and SNX12 (NM_001256188:c.470G>A; p.Arg157His), along with a variant in the TERT promoter (C250T, NM_198253.2: c.-146G>A), were already present in primary O2005. Allele frequencies of the 11 variants were calculated to assess their potential as putative driver genes. A missense change in NDST4 (NM_022569:c.2392C>G; p.Leu798Val) on 4q exhibited an increasing allele frequency (~ 20%, primary O2005, 80%, recurrent O2010 and 100%, xenograft1), consistent with a selection event. Sequencing of NDST4 in a cohort of 15 oligodendrogliomas, however, revealed no additional cases with potential protein disrupting variants. Our analysis illuminated a tumor evolutionary series of events, which included 1p/19q codeletion, IDH1 R132H, and TERT C250T as early events, followed by loss of function of NDST4 and mutations in FUBP1 and CIC as late events.
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Importance: Somatic mutations causing clonal expansion of hematopoietic cells (clonal hematopoiesis of indeterminate potential [CHIP]) are increased with age and associated with atherosclerosis and inflammation. Age and inflammation are the major risk factors for heart failure, yet the association of CHIP with heart failure in humans is unknown. Objective: To assess the potential prognostic significance of CHIP in patients with chronic heart failure (CHF) owing to ischemic origin. Design, Setting, and Participants: We analyzed bone marrow-derived mononuclear cells from 200 patients with CHF by deep targeted amplicon sequencing to detect the presence of CHIP and associated such with long-term prognosis in patients with CHF at University Hospital Frankfurt, Frankfurt, Germany. Data were analyzed between October 2017 and April 2018. Results: Median age of the patients was 65 years. Forty-seven mutations with a variant allele fraction (VAF) of at least 0.02 were found in 38 of 200 patients with CHF (18.5%). The somatic mutations most commonly occurred in the genes DNMT3A (14 patients), TET2 (9 patients), KDM6A (4 patients), and BCOR (3 patients). Patients with CHIP were older and more frequently had a history of hypertension. During a median follow-up of 4.4 years, a total of 53 patients died, and 23 patients required hospitalization for heart failure. There was a significantly worse long-term clinical outcome for patients with either DNMT3A or TET2 mutations compared with non-CHIP carriers. By multivariable Cox proportional regression analysis, the presence of somatic mutations within TET2 or DNMT3A (HR, 2.1; 95% CI, 1.1-4.0; P = .02, for death combined with heart failure hospitalization) and age (HR, 1.04; 95% CI, 1.01-1.07 per year; P = .005) but not a history of hypertension remained independently associated with adverse outcome. Importantly, there was a significant dose-response association between VAF and clinical outcome. Conclusions and Relevance: Our data suggest that somatic mutations in hematopoietic cells, specifically in the most commonly mutated CHIP driver genes TET2 and DNMT3A, may be significantly associated with the progression and poor prognosis of CHF. Future studies will have to validate our findings in larger cohorts and address whether targeting specific inflammatory pathways may be valuable for precision medicine in patients with CHF carrying specific mutations encoding for CHIP.
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Evolução Clonal/genética , Insuficiência Cardíaca/mortalidade , Hematopoese/genética , Isquemia Miocárdica/mortalidade , Idoso , Alelos , Aterosclerose/complicações , Aterosclerose/genética , Células da Medula Óssea , Doença Crônica , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Dioxigenases , Feminino , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Hospitalização , Humanos , Hipertensão , Inflamação/complicações , Inflamação/genética , Masculino , Pessoa de Meia-Idade , Monócitos , Mutação , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Prognóstico , Proteínas Proto-Oncogênicas/genética , Fatores de RiscoRESUMO
The original version of this Article contained an error in the Acknowledgements section.
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LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML. The conditional knockout led to an increased expression of multiple genes regulated by the important myeloid transcription factors GFI1 and PU.1. These include the transcription factors GFI1B and IRF8. We also compared the effect of different irreversible and reversible inhibitors of LSD1 in AML and could show that only tranylcypromine derivatives were capable of inducing a differentiation response. We employed a conditional knock-in model of inactive, mutant LSD1 to study the effect of only interfering with LSD1 enzymatic activity. While this was sufficient to initiate differentiation, it did not result in a survival benefit in mice. Hence, we believe that targeting both enzymatic and scaffolding functions of LSD1 is required to efficiently treat AML. This finding as well as the identified biomarkers may be relevant for the treatment of AML patients with LSD1 inhibitors.
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Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases/antagonistas & inibidores , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Tranilcipromina/farmacologia , Animais , Antidepressivos/farmacologia , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histona Desmetilases/fisiologia , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Clonal hematopoiesis of indeterminate potential (CHIP) is caused by recurrent somatic mutations leading to clonal blood cell expansion. However, direct evidence of the fitness of CHIP-mutated human hematopoietic stem cells (HSCs) in blood reconstitution is lacking. Because myeloablative treatment and transplantation enforce stress on HSCs, we followed 81 patients with solid tumors or lymphoid diseases undergoing autologous stem cell transplantation (ASCT) for the development of CHIP. We found a high incidence of CHIP (22%) after ASCT with a high mean variant allele frequency (VAF) of 10.7%. Most mutations were already present in the graft, albeit at lower VAFs, demonstrating a selective reconstitution advantage of mutated HSCs after ASCT. However, patients with CHIP mutations in DNA-damage response genes showed delayed neutrophil reconstitution. Thus, CHIP-mutated stem and progenitor cells largely gain on clone size upon ASCT-related blood reconstitution, leading to an increased future risk of CHIP-associated complications.
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Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Mutação , Neoplasias/genética , Neoplasias/terapia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transplante AutólogoRESUMO
IDH1R132H (isocitrate dehydrogenase 1) mutations play a key role in the development of low-grade gliomas. IDH1wt converts isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP+), whereas IDH1R132H uses α-ketoglutarate and NADPH to generate the oncometabolite 2-hydroxyglutarate (2-HG). While the effects of 2-HG have been the subject of intense research, the 2-HG independent effects of IDH1R132H are still ambiguous. The present study demonstrates that IDH1R132H expression but not 2-HG alone leads to significantly decreased tricarboxylic acid (TCA) cycle metabolites, reduced proliferation, and enhanced sensitivity to irradiation in both glioblastoma cells and astrocytes in vitro. Glioblastoma cells, but not astrocytes, showed decreased NADPH and NAD+ levels upon IDH1R132H transduction. However, in astrocytes IDH1R132H led to elevated expression of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT). These effects were not 2-HG mediated. This suggests that IDH1R132H cells utilize NAD+ to restore NADP pools, which only astrocytes could compensate via induction of NAMPT. We found that the expression of NAMPT is lower in patient-derived IDH1-mutant glioma cells and xenografts compared to IDH1-wildtype models. The Cancer Genome Atlas (TCGA) data analysis confirmed lower NAMPT expression in IDH1-mutant versus IDH1-wildtype gliomas. We show that the IDH1 mutation directly affects the energy homeostasis and redox state in a cell-type dependent manner. Targeting the impairments in metabolism and redox state might open up new avenues for treating IDH1-mutant gliomas.
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Autophagy is a well-described degradation mechanism that promotes cell survival upon nutrient starvation and other forms of cellular stresses. In addition, there is growing evidence showing that autophagy can exert a lethal function via autophagic cell death (ACD). As ACD has been implicated in apoptosis-resistant glioblastoma (GBM), there is a high medical need for identifying novel ACD-inducing drugs. Therefore, we screened a library containing 70 autophagy-inducing compounds to induce ATG5-dependent cell death in human MZ-54 GBM cells. Here, we identified three compounds, i.e. loperamide, pimozide, and STF-62247 that significantly induce cell death in several GBM cell lines compared to CRISPR/Cas9-generated ATG5- or ATG7-deficient cells, pointing to a death-promoting role of autophagy. Further cell death analyses conducted using pharmacological inhibitors revealed that apoptosis, ferroptosis, and necroptosis only play minor roles in loperamide-, pimozide- or STF-62247-induced cell death. Intriguingly, these three compounds induce massive lipidation of the autophagy marker protein LC3B as well as the formation of LC3B puncta, which are characteristic of autophagy. Furthermore, loperamide, pimozide, and STF-62247 enhance the autophagic flux in parental MZ-54 cells, but not in ATG5 or ATG7 knockout (KO) MZ-54 cells. In addition, loperamide- and pimozide-treated cells display a massive formation of autophagosomes and autolysosomes at the ultrastructural level. Finally, stimulation of autophagy by all three compounds is accompanied by dephosphorylation of mammalian target of rapamycin complex 1 (mTORC1), a well-known negative regulator of autophagy. In summary, our results indicate that loperamide, pimozide, and STF-62247 induce ATG5- and ATG7-dependent cell death in GBM cells, which is preceded by a massive induction of autophagy. These findings emphasize the lethal function and potential clinical relevance of hyperactivated autophagy in GBM.
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Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Loperamida/farmacologia , Pimozida/farmacologia , Piridinas/farmacologia , Tiazóis/farmacologia , Autofagossomos/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/ultraestrutura , Linhagem Celular Tumoral , Endossomos/metabolismo , Glioblastoma/patologia , Glioblastoma/ultraestrutura , Células HT29 , Humanos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismoRESUMO
Background: The evolution of primary glioblastoma (GBM) is poorly understood. Multifocal GBM (ie, multiple synchronous lesions in one patient) could elucidate GBM development. Methods: We present the first comprehensive study of 12 GBM foci from 6 patients using array-CGH, spectral karyotyping, gene expression arrays, and next-generation sequencing. Results: Multifocal GBMs genetically resemble primary GBMs. Comparing foci from the same patient proved their monoclonal origin. All tumors harbored alterations in the 3 GBM core pathways: RTK/PI3K, p53, and RB regulatory pathways with aberrations of EGFR and CDKN2A/B in all (100%) patients. This unexpected high frequency reflects a distinct genetic signature of multifocal GBMs and might account for their highly malignant and invasive phenotype. Surprisingly, the types of mutations in these genes/pathways were different in tumor foci from the same patients. For example, we found distinct mutations/aberrations in PTEN, TP53, EGFR, and CDKN2A/B, which therefore must have occurred independently and late during tumor development. We also identified chromothripsis as a late event and in tumors with wild-type TP53. Only 2 events were found to be early in all patients: single copy loss of PTEN and TERT promoter point mutations. Conclusions: Multifocal GBMs develop through parallel genetic evolution. The high frequency of alterations in 3 main pathways suggests that these are essential steps in GBM evolution; however, their late occurrence indicates that they are not founder events but rather subclonal drivers. This might account for the marked genetic heterogeneity seen in primary GBM and therefore has important implications for GBM therapy.
Assuntos
Neoplasias Encefálicas/genética , Evolução Clonal , Evolução Molecular , Glioblastoma/genética , Expressão Gênica , Humanos , Mutação , Transdução de SinaisAssuntos
Hematopoiese Clonal/genética , Insuficiência Cardíaca/genética , Doença Crônica , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Ecocardiografia , Frequência do Gene , Variação Genética , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/patologia , Humanos , Estimativa de Kaplan-Meier , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , PrognósticoRESUMO
Changes in gene expression programs play a central role in cancer. Chromosomal aberrations such as deletions, duplications and translocations of DNA segments can lead to highly significant positive correlations of gene expression levels of neighboring genes. This should be utilized to improve the analysis of tumor expression profiles. Here, we develop a novel model class of autoregressive higher-order Hidden Markov Models (HMMs) that carefully exploit local data-dependent chromosomal dependencies to improve the identification of differentially expressed genes in tumor. Autoregressive higher-order HMMs overcome generally existing limitations of standard first-order HMMs in the modeling of dependencies between genes in close chromosomal proximity by the simultaneous usage of higher-order state-transitions and autoregressive emissions as novel model features. We apply autoregressive higher-order HMMs to the analysis of breast cancer and glioma gene expression data and perform in-depth model evaluation studies. We find that autoregressive higher-order HMMs clearly improve the identification of overexpressed genes with underlying gene copy number duplications in breast cancer in comparison to mixture models, standard first- and higher-order HMMs, and other related methods. The performance benefit is attributed to the simultaneous usage of higher-order state-transitions in combination with autoregressive emissions. This benefit could not be reached by using each of these two features independently. We also find that autoregressive higher-order HMMs are better able to identify differentially expressed genes in tumors independent of the underlying gene copy number status in comparison to the majority of related methods. This is further supported by the identification of well-known and of previously unreported hotspots of differential expression in glioblastomas demonstrating the efficacy of autoregressive higher-order HMMs for the analysis of individual tumor expression profiles. Moreover, we reveal interesting novel details of systematic alterations of gene expression levels in known cancer signaling pathways distinguishing oligodendrogliomas, astrocytomas and glioblastomas. An implementation is available under www.jstacs.de/index.php/ARHMM.
Assuntos
Algoritmos , Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Glioma/genética , Cadeias de Markov , Modelos Teóricos , Biomarcadores Tumorais/genética , Aberrações Cromossômicas , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de SinaisRESUMO
Microgravity and cosmic rays as found in space are difficult to recreate on earth. However, ground-based models exist to simulate space flight experiments. In the present study, an experimental model was utilized to monitor gene expression changes in fetal skin fibroblasts of murine origin. Cells were continuously subjected for 65 h to a low dose (55 mSv) of ionizing radiation (IR), comprising a mixture of highlinear energy transfer (LET) neutrons and low-LET gamma-rays, and/or simulated microgravity using the random positioning machine (RPM), after which microarrays were performed. The data were analyzed both by gene set enrichment analysis (GSEA) and single gene analysis (SGA). Simulated microgravity affected fetal murine fibroblasts by inducing oxidative stress responsive genes. Three of these genes are targets of the nuclear factorerythroid 2 p45-related factor 2 (Nrf2), which may play a role in the cell response to simulated microgravity. In addition, simulated gravity decreased the expression of genes involved in cytoskeleton remodeling, which may have been caused by the downregulation of the serum response factor (SRF), possibly through the Rho signaling pathway. Similarly, chronic exposure to low-dose IR caused the downregulation of genes involved in cytoskeleton remodeling, as well as in cell cycle regulation and DNA damage response pathways. Many of the genes or gene sets that were altered in the individual treatments (RPM or IR) were not altered in the combined treatment (RPM and IR), indicating a complex interaction between RPM and IR.