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The recent acceleration of commercial, private and multi-national spaceflight has created an unprecedented level of activity in low Earth orbit, concomitant with the largest-ever number of crewed missions entering space and preparations for exploration-class (lasting longer than one year) missions. Such rapid advancement into space from many new companies, countries and space-related entities has enabled a 'second space age'. This era is also poised to leverage, for the first time, modern tools and methods of molecular biology and precision medicine, thus enabling precision aerospace medicine for the crews. The applications of these biomedical technologies and algorithms are diverse, and encompass multi-omic, single-cell and spatial biology tools to investigate human and microbial responses to spaceflight. Additionally, they extend to the development of new imaging techniques, real-time cognitive assessments, physiological monitoring and personalized risk profiles tailored for astronauts. Furthermore, these technologies enable advancements in pharmacogenomics, as well as the identification of novel spaceflight biomarkers and the development of corresponding countermeasures. In this Perspective, we highlight some of the recent biomedical research from the National Aeronautics and Space Administration, Japan Aerospace Exploration Agency, European Space Agency and other space agencies, and detail the entrance of the commercial spaceflight sector (including SpaceX, Blue Origin, Axiom and Sierra Space) into aerospace medicine and space biology, the first aerospace medicine biobank, and various upcoming missions that will utilize these tools to ensure a permanent human presence beyond low Earth orbit, venturing out to other planets and moons.
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How hematopoietic stem cells (HSCs) coordinate the regulation of opposing cellular mechanisms such as self-renewal and differentiation commitment remains unclear. Here we identified the transcription factor and chromatin remodeler Satb1 as a critical regulator of HSC fate. HSCs lacking Satb1 had defective self-renewal, were less quiescent and showed accelerated lineage commitment, which resulted in progressive depletion of functional HSCs. The enhanced commitment was caused by less symmetric self-renewal and more symmetric differentiation divisions of Satb1-deficient HSCs. Satb1 simultaneously repressed sets of genes encoding molecules involved in HSC activation and cellular polarity, including Numb and Myc, which encode two key factors for the specification of stem-cell fate. Thus, Satb1 is a regulator that promotes HSC quiescence and represses lineage commitment.
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Células-Tronco Hematopoéticas/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Polaridade Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Chronic natural killer large granular lymphocyte (NK-LGL) leukemia, also referred to as chronic lymphoproliferative disorder of NK cells, is a rare disorder defined by prolonged expansion of clonal NK cells. Similar prevalence of STAT3 mutations in chronic T-LGL and NK-LGL leukemia is suggestive of common pathogenesis. We undertook whole-genome sequencing to identify mutations unique to NK-LGL leukemia. The results were analyzed to develop a resequencing panel that was applied to 58 patients. Phosphatidylinositol 3-kinase pathway gene mutations (PIK3CD/PIK3AP1) and TNFAIP3 mutations were seen in 5% and 10% of patients, respectively. TET2 was exceptional in that mutations were present in 16 (28%) of 58 patient samples, with evidence that TET2 mutations can be dominant and exclusive to the NK compartment. Reduced-representation bisulfite sequencing revealed that methylation patterns were significantly altered in TET2 mutant samples. The promoter of TET2 and that of PTPRD, a negative regulator of STAT3, were found to be methylated in additional cohort samples, largely confined to the TET2 mutant group. Mutations in STAT3 were observed in 19 (33%) of 58 patient samples, 7 of which had concurrent TET2 mutations. Thrombocytopenia and resistance to immunosuppressive agents were uniquely observed in those patients with only TET2 mutation (Games-Howell post hoc test, P = .0074; Fisher's exact test, P = .00466). Patients with STAT3 mutation, inclusive of those with TET2 comutation, had lower hematocrit, hemoglobin, and absolute neutrophil count compared with STAT3 wild-type patients (Welch's t test, P ≤ .015). We present the discovery of TET2 mutations in chronic NK-LGL leukemia and evidence that it identifies a unique molecular subtype.
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Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Leucemia Linfocítica Granular Grande/genética , Mutação , Proteínas de Neoplasias/genética , Sistema de Registros , Doença Crônica , Proteínas de Ligação a DNA/sangue , Dioxigenases/sangue , Feminino , Humanos , Leucemia Linfocítica Granular Grande/sangue , Masculino , Proteínas de Neoplasias/sangueRESUMO
Recent studies have reported that activation-induced cytidine deaminase (AID) and ten-eleven-translocation (TET) family members regulate active DNA demethylation. Genetic alterations of TET2 occur in myeloid malignancies, and hematopoietic-specific loss of Tet2 induces aberrant hematopoietic stem cell (HSC) self-renewal/differentiation, implicating TET2 as a master regulator of normal and malignant hematopoiesis. Despite the functional link between AID and TET in epigenetic gene regulation, the role of AID loss in hematopoiesis and myeloid transformation remains to be investigated. Here, we show that Aid loss in mice leads to expansion of myeloid cells and reduced erythroid progenitors resulting in anemia, with dysregulated expression of Cebpa and Gata1, myeloid/erythroid lineage-specific transcription factors. Consistent with data in the murine context, silencing of AID in human bone marrow cells skews differentiation toward myelomonocytic lineage. However, in contrast to Tet2 loss, Aid loss does not contribute to enhanced HSC self-renewal or cooperate with Flt3-ITD to induce myeloid transformation. Genome-wide transcription and differential methylation analysis uncover the critical role of Aid as a key epigenetic regulator. These results indicate that AID and TET2 share common effects on myeloid and erythroid lineage differentiation, however, their role is nonredundant in regulating HSC self-renewal and in myeloid transformation.
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Diferenciação Celular , Citidina Desaminase/fisiologia , Metilação de DNA , Células-Tronco Hematopoéticas/metabolismo , Animais , Linhagem da Célula , Autorrenovação Celular , Transformação Celular Neoplásica , Citidina Desaminase/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Dioxigenases , Células Eritroides/citologia , Inativação Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Células Mieloides/citologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologiaRESUMO
We have developed an enhanced form of reduced representation bisulfite sequencing with extended genomic coverage, which resulted in greater capture of DNA methylation information of regions lying outside of traditional CpG islands. Applying this method to primary human bone marrow specimens from patients with Acute Myelogeneous Leukemia (AML), we demonstrated that genetically distinct AML subtypes display diametrically opposed DNA methylation patterns. As compared to normal controls, we observed widespread hypermethylation in IDH mutant AMLs, preferentially targeting promoter regions and CpG islands neighboring the transcription start sites of genes. In contrast, AMLs harboring translocations affecting the MLL gene displayed extensive loss of methylation of an almost mutually exclusive set of CpGs, which instead affected introns and distal intergenic CpG islands and shores. When analyzed in conjunction with gene expression profiles, it became apparent that these specific patterns of DNA methylation result in differing roles in gene expression regulation. However, despite this subtype-specific DNA methylation patterning, a much smaller set of CpG sites are consistently affected in both AML subtypes. Most CpG sites in this common core of aberrantly methylated CpGs were hypermethylated in both AML subtypes. Therefore, aberrant DNA methylation patterns in AML do not occur in a stereotypical manner but rather are highly specific and associated with specific driving genetic lesions.
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Metilação de DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Sequência de Bases , Ilhas de CpG/genética , Genoma Humano , Células HCT116 , Histona-Lisina N-Metiltransferase , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
Microgravity is associated with immunological dysfunction, though the mechanisms are poorly understood. Here, using single-cell analysis of human peripheral blood mononuclear cells (PBMCs) exposed to short term (25 hours) simulated microgravity, we characterize altered genes and pathways at basal and stimulated states with a Toll-like Receptor-7/8 agonist. We validate single-cell analysis by RNA sequencing and super-resolution microscopy, and against data from the Inspiration-4 (I4) mission, JAXA (Cell-Free Epigenome) mission, Twins study, and spleens from mice on the International Space Station. Overall, microgravity alters specific pathways for optimal immunity, including the cytoskeleton, interferon signaling, pyroptosis, temperature-shock, innate inflammation (e.g., Coronavirus pathogenesis pathway and IL-6 signaling), nuclear receptors, and sirtuin signaling. Microgravity directs monocyte inflammatory parameters, and impairs T cell and NK cell functionality. Using machine learning, we identify numerous compounds linking microgravity to immune cell transcription, and demonstrate that the flavonol, quercetin, can reverse most abnormal pathways. These results define immune cell alterations in microgravity, and provide opportunities for countermeasures to maintain normal immunity in space.
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Leucócitos Mononucleares , Análise de Célula Única , Voo Espacial , Simulação de Ausência de Peso , Animais , Feminino , Humanos , Masculino , Camundongos , Imunidade Inata , Inflamação/imunologia , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Aprendizado de Máquina , Camundongos Endogâmicos C57BL , Quercetina/farmacologia , Transdução de Sinais , Linfócitos T/imunologia , Ausência de PesoRESUMO
BACKGROUND: DNA methylation profiling reveals important differentially methylated regions (DMRs) of the genome that are altered during development or that are perturbed by disease. To date, few programs exist for regional analysis of enriched or whole-genome bisulfate conversion sequencing data, even though such data are increasingly common. Here, we describe an open-source, optimized method for determining empirically based DMRs (eDMR) from high-throughput sequence data that is applicable to enriched whole-genome methylation profiling datasets, as well as other globally enriched epigenetic modification data. RESULTS: Here we show that our bimodal distribution model and weighted cost function for optimized regional methylation analysis provides accurate boundaries of regions harboring significant epigenetic modifications. Our algorithm takes the spatial distribution of CpGs into account for the enrichment assay, allowing for optimization of the definition of empirical regions for differential methylation. Combined with the dependent adjustment for regional p-value combination and DMR annotation, we provide a method that may be applied to a variety of datasets for rapid DMR analysis. Our method classifies both the directionality of DMRs and their genome-wide distribution, and we have observed that shows clinical relevance through correct stratification of two Acute Myeloid Leukemia (AML) tumor sub-types. CONCLUSIONS: Our weighted optimization algorithm eDMR for calling DMRs extends an established DMR R pipeline (methylKit) and provides a needed resource in epigenomics. Our method enables an accurate and scalable way of finding DMRs in high-throughput methylation sequencing experiments. eDMR is available for download at http://code.google.com/p/edmr/.
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Algoritmos , Metilação de DNA , Anotação de Sequência Molecular/métodos , Ilhas de CpG , Epigenômica/métodos , Genoma , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia/genética , Análise de Sequência de DNARESUMO
Introduction: Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and dysplasia. The gene encoding ten-eleven translocation 2 (tet2), a dioxygenase enzyme that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, is a recurrently mutated tumor suppressor gene in MDS and other myeloid malignancies. Previously, we reported a stable zebrafish line with a loss-of-function mutation in the tet2 gene. The tet2m/m-mutant zebrafish developed a pre-MDS state with kidney marrow dysplasia, but normal circulating blood counts by 11 months of age and accompanying anemia, signifying the onset of MDS, by 24 months of age. Methods: In the current study, we collected progenitor cells from the kidney marrows of the adult tet2m/m and tet2wt/wt fish at 4 and 15 months of age and conducted enhanced reduced representation of bisulfite sequencing (ERRBS) and bulk RNA-seq to measure changes in DNA methylation and gene expression of hematopoietic stem and progenitor cells (HSPCs). Results and discussion: A global increase in DNA methylation of gene promoter regions and CpG islands was observed in tet2m/m HSPCs at 4 months of age when compared with the wild type. Furthermore, hypermethylated genes were significantly enriched for targets of SUZ12 and the metal-response-element-binding transcription factor 2 (MTF2)-involved in the polycomb repressive complex 2 (PRC2). However, between 4 and 15 months of age, we observed a paradoxical global decrease in DNA methylation in tet2m/m HSPCs. Gene expression analyses identified upregulation of genes associated with mTORC1 signaling and interferon gamma and alpha responses in tet2m/m HSPCs at 4 months of age when compared with the wild type. Downregulated genes in HSPCs of tet2-mutant fish at 4 months of age were enriched for cell cycle regulation, heme metabolism, and interleukin 2 (IL2)/signal transducer and activator of transcription 5 (STAT5) signaling, possibly related to increased self-renewal and clonal advantage in HSPCs with tet2 loss of function. Finally, there was an overall inverse correlation between overall increased promoter methylation and gene expression.
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Despite the prognostic value of arterial stiffness (AS) and pulsatile hemodynamics (PH) for cardiovascular morbidity and mortality, epigenetic modifications that contribute to AS/PH remain unknown. To gain a better understanding of the link between epigenetics (DNA methylation) and AS/PH, we examined the relationship of eight measures of AS/PH with CpG sites and co-methylated regions using multi-ancestry participants from Trans-Omics for Precision Medicine (TOPMed) Multi-Ethnic Study of Atherosclerosis (MESA) with sample sizes ranging from 438 to 874. Epigenome-wide association analysis identified one genome-wide significant CpG (cg20711926-CYP1B1) associated with aortic augmentation index (AIx). Follow-up analyses, including gene set enrichment analysis, expression quantitative trait methylation analysis, and functional enrichment analysis on differentially methylated positions and regions, further prioritized three CpGs and their annotated genes (cg23800023-ETS1, cg08426368-TGFB3, and cg17350632-HLA-DPB1) for AIx. Among these, ETS1 and TGFB3 have been previously prioritized as candidate genes. Furthermore, both ETS1 and HLA-DPB1 have significant tissue correlations between Whole Blood and Aorta in GTEx, which suggests ETS1 and HLA-DPB1 could be potential biomarkers in understanding pathophysiology of AS/PH. Overall, our findings support the possible role of epigenetic regulation via DNA methylation of specific genes associated with AIx as well as identifying potential targets for regulation of AS/PH.
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Aterosclerose , Epigênese Genética , Humanos , Epigenoma , Fator de Crescimento Transformador beta3/genética , Medicina de Precisão , Estudo de Associação Genômica Ampla , Metilação de DNA , Ilhas de CpG/genética , Aterosclerose/genéticaRESUMO
Despite the prognostic value of arterial stiffness (AS) and pulsatile hemodynamics (PH) for cardiovascular morbidity and mortality, epigenetic modifications that contribute to AS/PH remain unknown. To gain a better understanding of the link between epigenetics (DNA methylation) and AS/PH, we examined the relationship of eight measures of AS/PH with CpG sites and co-methylated regions using multi-ancestry participants from Trans-Omics for Precision Medicine (TOPMed) Multi-Ethnic Study of Atherosclerosis (MESA) with sample sizes ranging from 438 to 874. Epigenome-wide association analysis identified one genome-wide significant CpG (cg20711926-CYP1B1) associated with aortic augmentation index (AIx). Follow-up analyses, including gene set enrichment analysis, expression quantitative trait methylation analysis, and functional enrichment analysis on differentially methylated positions and regions, further prioritized three CpGs and their annotated genes (cg23800023-ETS1, cg08426368-TGFB3, and cg17350632-HLA-DPB1) for AIx. Among these, ETS1 and TGFB3 have been previously prioritized as candidate genes. Furthermore, both ETS1 and HLA-DPB1 have significant tissue correlations between Whole Blood and Aorta in GTEx, which suggests ETS1 and HLA-DPB1 could be potential biomarkers in understanding pathophysiology of AS/PH. Overall, our findings support the possible role of epigenetic regulation via DNA methylation of specific genes associated with AIx as well as identifying potential targets for regulation of AS/PH.
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Acute myeloid leukemia (AML) is an aggressive disease with complex and heterogeneous biology. Although several genomic classifications have been proposed, there is a growing interest in going beyond genomics to stratify AML. In this study, we profile the sphingolipid family of bioactive molecules in 213 primary AML samples and 30 common human AML cell lines. Using an integrative approach, we identify two distinct sphingolipid subtypes in AML characterized by a reciprocal abundance of hexosylceramide (Hex) and sphingomyelin (SM) species. The two Hex-SM clusters organize diverse samples more robustly than known AML driver mutations and are coupled to latent transcriptional states. Using transcriptomic data, we develop a machine-learning classifier to infer the Hex-SM status of AML cases in TCGA and BeatAML clinical repositories. The analyses show that the sphingolipid subtype with deficient Hex and abundant SM is enriched for leukemic stemness transcriptional programs and comprises an unappreciated high-risk subgroup with poor clinical outcomes. Our sphingolipid-focused examination of AML identifies patients least likely to benefit from standard of care and raises the possibility that sphingolipidomic interventions could switch the subtype of AML patients who otherwise lack targetable alternatives.
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Acute myeloid leukemia patients with FMS-like tyrosine kinase 3-internal tandem duplications and mixed lineage leukemia-protein AF9 fusion proteins suffer from poor clinical outcomes. The MOLM-13 acute myeloid leukemia cell line harbors both of these abnormalities and is used in CRISPR experiments to identify disease drivers. However, experimental observations may be biased or inconclusive in the absence of experimentally validated positive control genes. We validated sgRNAs for knockdown of TP53 for cell proliferation and for DCK knockdown and CDA upregulation for cytarabine resistance control genes in MOLM-13 cells. We have provided a detailed CRISPR protocol applicable to both gene knockdown or activation experiments and downstream leukemic phenotype analyses. Inclusion of these controls in CRISPR experiments will enhance the capacity to identify novel myeloid leukemia drivers in MOLM-13 cells.
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Citarabina , Leucemia Mieloide Aguda , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citarabina/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genéticaRESUMO
Measurable residual disease (MRD) assessment provides a potent indicator of the efficacy of anti-leukemic therapy. It is unknown, however, whether integrating MRD with molecular profiling better identifies patients at risk of relapse. To investigate the clinical relevance of MRD in relation to a molecular-based prognostic schema, we measured MRD by flow cytometry in 189 AML patients enrolled in ECOG-ACRIN E1900 trial (NCT00049517) in morphologic complete remission (CR) (28.8 % of the original cohort) representing 44.4 % of CR patients. MRD positivity was defined as ≥ 0.1 % of leukemic bone marrow cells. Risk classification was based on standard cytogenetics, fluorescence-in-situ-hybridization, somatic gene analysis, and sparse whole genome sequencing for copy number ascertainment. At 84.6 months median follow-up of patients still alive at the time of analysis (range 47.0-120 months), multivariate analysis demonstrated that MRD status at CR (p = 0.001) and integrated molecular risk (p = 0.0004) independently predicted overall survival (OS). Among risk classes, MRD status significantly affected OS only in the favorable risk group (p = 0.002). Expression of CD25 (α-chain of the interleukin-2 receptor) by leukemic myeloblasts at diagnosis negatively affected OS independent of post-treatment MRD levels. These data suggest that integrating MRD with genetic profiling and pre-treatment CD25 expression may improve prognostication in AML.
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Leucemia Mieloide Aguda , Humanos , Citometria de Fluxo , Genômica , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Neoplasia Residual/genética , Neoplasia Residual/diagnóstico , PrognósticoRESUMO
Acute myeloid leukemia (AML) is an aggressive, heterogenous malignancy characterized by clonal expansion of bone marrow-derived myeloid progenitor cells. While our current understanding of the molecular and genomic landscape of AML has evolved dramatically and opened avenues for molecularly targeted therapeutics to improve upon standard intensive induction chemotherapy, curative treatments are elusive, particularly in older patients. Responses to current AML treatments are transient and incomplete, necessitating the development of novel treatment strategies to improve outcomes. To this end, harnessing the power of bioactive sphingolipids to treat cancer shows great promise. Sphingolipids are involved in many hallmarks of cancer of paramount importance in AML. Leukemic blast survival is influenced by cellular levels of ceramide, a bona fide pro-death molecule, and its conversion to signaling molecules such as sphingosine-1-phosphate and glycosphingolipids. Preclinical studies demonstrate the efficacy of therapeutics that target dysregulated sphingolipid metabolism as well as their combinatorial synergy with clinically-relevant therapeutics. Thus, increased understanding of sphingolipid dysregulation may be exploited to improve AML patient care and outcomes. This review summarizes the current knowledge of dysregulated sphingolipid metabolism in AML, evaluates how pro-survival sphingolipids promote AML pathogenesis, and discusses the therapeutic potential of targeting these dysregulated sphingolipid pathways.
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Leucemia Mieloide Aguda , Esfingolipídeos , Idoso , Ceramidas/metabolismo , Ceramidas/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/terapia , Transdução de Sinais , Esfingolipídeos/metabolismo , Esfingolipídeos/uso terapêuticoRESUMO
Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.
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Leucemia Mieloide Aguda , Enzimas de Conjugação de Ubiquitina , Proliferação de Células/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Oncogenes , Transdução de Sinais/genética , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors.
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Neoplasias da Mama/genética , Metilação de DNA , Epigênese Genética , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica , Estudos de Coortes , Ilhas de CpG/genética , Replicação do DNA/genética , Feminino , Genoma Humano/genética , Instabilidade Genômica/genética , Genômica/métodos , Humanos , Células MCF-7 , Mutação , Regiões Promotoras Genéticas/genética , Análise de SobrevidaRESUMO
Patients with acute myeloid leukemia (AML) frequently relapse after chemotherapy, yet the mechanism by which AML reemerges is not fully understood. Herein, we show that primary AML cells enter a senescence-like phenotype following chemotherapy in vitro and in vivo. This is accompanied by induction of senescence/inflammatory and embryonic diapause transcriptional programs, with downregulation of MYC and leukemia stem cell genes. Single-cell RNA sequencing suggested depletion of leukemia stem cells in vitro and in vivo, and enrichment for subpopulations with distinct senescence-like cells. This senescence effect was transient and conferred superior colony-forming and engraftment potential. Entry into this senescence-like phenotype was dependent on ATR, and persistence of AML cells was severely impaired by ATR inhibitors. Altogether, we propose that AML relapse is facilitated by a senescence-like resilience phenotype that occurs regardless of their stem cell status. Upon recovery, these post-senescence AML cells give rise to relapsed AMLs with increased stem cell potential. SIGNIFICANCE: Despite entering complete remission after chemotherapy, relapse occurs in many patients with AML. Thus, there is an urgent need to understand the relapse mechanism in AML and the development of targeted treatments to improve outcome. Here, we identified a senescence-like resilience phenotype through which AML cells can survive and repopulate leukemia.This article is highlighted in the In This Issue feature, p. 1307.
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Leucemia Mieloide Aguda/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Células-Tronco Neoplásicas/citologia , Indução de Remissão , Animais , Linhagem Celular Tumoral/citologia , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Recidiva Local de Neoplasia/patologia , FenótipoRESUMO
Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory "dimensions" coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module.
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A key challenge in epigenetics is to determine the biological significance of epigenetic variation among individuals. We present Coordinate Covariation Analysis (COCOA), a computational framework that uses covariation of epigenetic signals across individuals and a database of region sets to annotate epigenetic heterogeneity. COCOA is the first such tool for DNA methylation data and can also analyze any epigenetic signal with genomic coordinates. We demonstrate COCOA's utility by analyzing DNA methylation, ATAC-seq, and multi-omic data in supervised and unsupervised analyses, showing that COCOA provides new understanding of inter-sample epigenetic variation. COCOA is available on Bioconductor ( http://bioconductor.org/packages/COCOA ).
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Epigênese Genética , Epigenômica/métodos , Heterogeneidade Genética , Software , Neoplasias da Mama/genética , Metilação de DNA , Humanos , Anotação de Sequência MolecularRESUMO
Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations.