RESUMO
Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations, maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However, patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here, we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model, we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity.
Assuntos
Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Células-Tronco/metabolismo , Células-Tronco Neoplásicas/metabolismoRESUMO
Tumor-specific MHC class II (tsMHC-II) expression impacts tumor microenvironmental immunity. tsMHC-II positive cancer cells may act as surrogate antigen-presenting cells and targets for CD4+ T cell-mediated lysis. In colorectal cancer, tsMHC-II negativity is common, in cell lines due to CIITA promoter methylation. To clarify mechanisms of tsMHC-II repression in colorectal cancer, we analyzed colorectal cancer organoids which are epigenetically faithful to tissue of origin. 15 primary colorectal cancer organoids were treated with IFNγ ± epigenetic modifiers: flow cytometry was used for tsMHC-II expression. qRT-PCR, total RNA sequencing, nanopore sequencing, bisulfite conversion/pyrosequencing, and Western blotting was used to quantitate CIITA, STAT1, IRF1, and JAK1 expression, mutations and promoter methylation and chromatin immunoprecipitation to quantitate H3K9ac, H3K9Me2, and EZH2 occupancy at CIITA. We define three types of response to IFNγ in colorectal cancer: strong, weak, and noninducibility. Delayed and restricted expression even with prolonged IFNγ exposure was due to IFNγ-mediated EZH2 occupancy at CIITA. tsMHC-II expression was enhanced by EZH2 and histone deacetylase inhibition in the weakly inducible organoids. Noninducibility is seen in three consensus molecular subtype 1 (CMS1) organoids due to JAK1 mutation. No organoid demonstrates CIITA promoter methylation. Providing IFNγ signaling is intact, most colorectal cancer organoids are class II inducible. Upregulation of tsMHC-II through targeted epigenetic therapy is seen in one of fifteen organoids. Our approach can serve as a blueprint for investigating the heterogeneity of specific epigenetic mechanisms of immune suppression across individual patients in other cancers and how these might be targeted to inform the conduct of future trials of epigenetic therapies as immune adjuvants more strategically in cancer. Significance: Cancer cell expression of MHC class II significantly impacts tumor microenvironmental immunity. Previous studies investigating mechanisms of repression of IFNγ-inducible class II expression using cell lines demonstrate epigenetic silencing of IFN pathway genes as a frequent immune evasion strategy. Unlike cell lines, patient-derived organoids maintain epigenetic fidelity to tissue of origin. In the first such study, we analyze patterns, dynamics, and epigenetic control of IFNγ-induced class II expression in a series of colorectal cancer organoids.
Assuntos
Neoplasias Colorretais , Genes MHC da Classe II , Humanos , Interferon gama/farmacologia , Metilação , Linhagem Celular , Neoplasias Colorretais/genéticaRESUMO
Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy caused by mutations in genes encoding transcriptional and epigenetic regulators together with signaling genes. It is characterized by a disturbance of differentiation and abnormal proliferation of hematopoietic progenitors. We have previously shown that each AML subtype establishes its own core gene regulatory network (GRN), consisting of transcription factors binding to their target genes and imposing a specific gene expression pattern that is required for AML maintenance. In this study, we integrate gene expression, open chromatin and ChIP data with promoter-capture Hi-C data to define a refined core GRN common to all patients with CEBPA-double mutant (CEBPAN/C) AML. These mutations disrupt the structure of a major regulator of myelopoiesis. We identify the binding sites of mutated C/EBPα proteins in primary cells, we show that C/EBPα, AP-1 factors and RUNX1 colocalize and are required for AML maintenance, and we employ single cell experiments to link important network nodes to the specific differentiation trajectory from leukemic stem to blast cells. Taken together, our study provides an important resource which predicts the specific therapeutic vulnerabilities of this AML subtype in human cells.
Assuntos
Redes Reguladoras de Genes , Leucemia Mieloide Aguda , Humanos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Mutação , Diferenciação Celular/genética , Leucemia Mieloide Aguda/patologiaRESUMO
The fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.
Assuntos
Proteína de Leucina Linfoide-Mieloide , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Epigênese Genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Genes Reguladores , CromatinaRESUMO
In an effort to identify novel drugs targeting fusion-oncogene-induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/metabolismo , Fosfolipase C gama/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Animais , Autorrenovação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteínas de Fusão Oncogênica/genética , Fosfolipase C gama/genética , Proteoma , Proteína 1 Parceira de Translocação de RUNX1/genética , Transcriptoma , Translocação GenéticaRESUMO
OBJECTIVES: Rapid, high throughput diagnostics are a valuable tool, allowing the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations so as to identify and isolate people with asymptomatic and symptomatic infections. Reagent shortages and restricted access to high throughput testing solutions have limited the effectiveness of conventional assays such as quantitative RT-PCR (RT-qPCR), particularly throughout the first months of the coronavirus disease 2019 pandemic. We investigated the use of LamPORE, where loop-mediated isothermal amplification (LAMP) is coupled to nanopore sequencing technology, for the detection of SARS-CoV-2 in symptomatic and asymptomatic populations. METHODS: In an asymptomatic prospective cohort, for 3 weeks in September 2020, health-care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self-swabbed with nasopharyngeal swabs weekly and supplied a saliva specimen daily. These samples were tested for SARS-CoV-2 RNA using the Oxford Nanopore LamPORE system and a reference RT-qPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza-like illness from March 2020 to June 2020 were similarly tested from nasopharyngeal swabs. RESULTS: In the asymptomatic cohort a total of 1200 participants supplied 23 427 samples (3966 swab, 19 461 saliva) over a 3-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% (decreasing to approximately 98% when clustered estimation was used) in both swab and saliva asymptomatic samples when compared with the reference RT-qPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%. CONCLUSIONS: LamPORE is a highly accurate methodology for the detection of SARS-CoV-2 in both symptomatic and asymptomatic population settings and can be used as an alternative to RT-qPCR.
Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , SARS-CoV-2/isolamento & purificação , COVID-19/virologia , Estudos de Coortes , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Limite de Detecção , Sequenciamento por Nanoporos , Nasofaringe/virologia , Poliproteínas/genética , Estudos Prospectivos , Reprodutibilidade dos Testes , Estudos Retrospectivos , SARS-CoV-2/genética , Saliva/virologia , Sensibilidade e Especificidade , Proteínas Virais/genéticaRESUMO
Acute myeloid leukemia development occurs in a stepwise fashion whereby an original driver mutation is followed by additional mutations. The first type of mutations tends to be in genes encoding members of the epigenetic/transcription regulatory machinery (i.e., RUNX1, DNMT3A, TET2), while the secondary mutations often involve genes encoding members of signaling pathways that cause uncontrolled growth of such cells such as the growth factor receptors c-KIT of FLT3. Patients usually present with both types of mutations, but it is currently unclear how both mutational events shape the epigenome in developing AML cells. To this end we generated an in vitro model of t(8;21) AML by expressing its driver oncoprotein RUNX1-ETO with or without a mutated (N822K) KIT protein. Expression of N822K-c-KIT strongly increases the self-renewal capacity of RUNX1-ETO-expressing cells. Global analysis of gene expression changes and alterations in the epigenome revealed that N822K-c-KIT expression profoundly influences the open chromatin landscape and transcription factor binding. However, our experiments also revealed that double mutant cells still differ from their patient-derived counterparts, highlighting the importance of studying patient cells to obtain a true picture of how gene regulatory networks have been reprogrammed during tumorigenesis.
Assuntos
Cromatina/metabolismo , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Leucemia Mieloide Aguda , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Transcrição Gênica , Translocação Genética , Substituição de Aminoácidos , Cromatina/patologia , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 21/metabolismo , Cromossomos Humanos Par 8/genética , Cromossomos Humanos Par 8/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feminino , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Proteínas Proto-Oncogênicas c-kit/genética , Proteína 1 Parceira de Translocação de RUNX1/genéticaRESUMO
Acute myeloid leukemia (AML) is associated with mutations in transcriptional and epigenetic regulator genes impairing myeloid differentiation. The t(8;21)(q22;q22) translocation generates the RUNX1-ETO fusion protein, which interferes with the hematopoietic master regulator RUNX1. We previously showed that the maintenance of t(8;21) AML is dependent on RUNX1-ETO expression. Its depletion causes extensive changes in transcription factor binding, as well as gene expression, and initiates myeloid differentiation. However, how these processes are connected within a gene regulatory network is unclear. To address this question, we performed Promoter-Capture Hi-C assays, with or without RUNX1-ETO depletion and assigned interacting cis-regulatory elements to their respective genes. To construct a RUNX1-ETO-dependent gene regulatory network maintaining AML, we integrated cis-regulatory element interactions with gene expression and transcription factor binding data. This analysis shows that RUNX1-ETO participates in cis-regulatory element interactions. However, differential interactions following RUNX1-ETO depletion are driven by alterations in the binding of RUNX1-ETO-regulated transcription factors.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Subunidade alfa 2 de Fator de Ligação ao Core , Elementos Facilitadores Genéticos , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda , Proteínas de Fusão Oncogênica , Regiões Promotoras Genéticas , Proteína 1 Parceira de Translocação de RUNX1 , Fator de Transcrição AP-1 , Translocação Genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 21/metabolismo , Cromossomos Humanos Par 8/genética , Cromossomos Humanos Par 8/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Deleção de Genes , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/genética , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Acute myeloid leukemia (AML) is a heterogeneous disease caused by a variety of alterations in transcription factors, epigenetic regulators and signaling molecules. To determine how different mutant regulators establish AML subtype-specific transcriptional networks, we performed a comprehensive global analysis of cis-regulatory element activity and interaction, transcription factor occupancy and gene expression patterns in purified leukemic blast cells. Here, we focused on specific subgroups of subjects carrying mutations in genes encoding transcription factors (RUNX1, CEBPα), signaling molecules (FTL3-ITD, RAS) and the nuclear protein NPM1). Integrated analysis of these data demonstrates that each mutant regulator establishes a specific transcriptional and signaling network unrelated to that seen in normal cells, sustaining the expression of unique sets of genes required for AML growth and maintenance.
Assuntos
Regulação Leucêmica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Leucemia Mieloide Aguda/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Transdução de Sinais/genética , Fatores de Transcrição/genética , Adulto JovemRESUMO
Oncogenic transcription factors such as the leukemic fusion protein RUNX1/ETO, which drives t(8;21) acute myeloid leukemia (AML), constitute cancer-specific but highly challenging therapeutic targets. We used epigenomic profiling data for an RNAi screen to interrogate the transcriptional network maintaining t(8;21) AML. This strategy identified Cyclin D2 (CCND2) as a crucial transmitter of RUNX1/ETO-driven leukemic propagation. RUNX1/ETO cooperates with AP-1 to drive CCND2 expression. Knockdown or pharmacological inhibition of CCND2 by an approved drug significantly impairs leukemic expansion of patient-derived AML cells and engraftment in immunodeficient murine hosts. Our data demonstrate that RUNX1/ETO maintains leukemia by promoting cell cycle progression and identifies G1 CCND-CDK complexes as promising therapeutic targets for treatment of RUNX1/ETO-driven AML.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Ciclina D2/genética , Animais , Linhagem Celular Tumoral , Cromossomos Humanos Par 21/genética , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Proteínas de Fusão Oncogênica/genética , Oncogenes/genética , Translocação Genética/genéticaRESUMO
Acute myeloid leukemia (AML) is a heterogeneous disease caused by recurrent mutations in the transcription regulatory machinery, resulting in abnormal growth and a block in differentiation. One type of recurrent mutations affects RUNX1, which is subject to mutations and translocations, the latter giving rise to fusion proteins with aberrant transcriptional activities. We recently compared the mechanism by which the products of the t(8;21) and the t(3;21) translocation RUNX1-ETO and RUNX1-EVI1 reprogram the epigenome. We demonstrated that a main component of the block in differentiation in both types of AML is direct repression of the gene encoding the myeloid regulator C/EBPα by both fusion proteins. Here, we examined at the global level whether C/EBPα is able to reverse aberrant chromatin programming in t(8;21) and t(3;21) AML. C/EBPα overexpression does not change oncoprotein expression or globally displace these proteins from their binding sites. Instead, it upregulates a core set of common target genes important for myeloid differentiation and represses genes regulating leukemia maintenance. This study, therefore, identifies common CEBPA-regulated pathways as targets for therapeutic intervention.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Reprogramação Celular , Epigênese Genética , Leucemia Mieloide Aguda/genética , Fatores de Transcrição , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/genética , Células Cultivadas , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Humanos , Leucemia Mieloide Aguda/patologia , MutaçãoRESUMO
Understanding and blocking the self-renewal pathway of preleukemia stem cells could prevent acute myeloid leukemia (AML) relapse. In this study, we show that increased FOXO1 represents a critical mechanism driving aberrant self-renewal in preleukemic cells expressing the t(8;21)-associated oncogene AML1-ETO (AE). Although generally considered as a tumor suppressor, FOXO1 is consistently upregulated in t(8;21) AML. Expression of FOXO1 in human CD34+ cells promotes a preleukemic state with enhanced self-renewal and dysregulated differentiation. The DNA binding domain of FOXO1 is essential for these functions. FOXO1 activates a stem cell molecular signature that is also present in AE preleukemia cells and preserved in t(8;21) patient samples. Genome-wide binding studies show that AE and FOXO1 share the majority of their binding sites, whereby FOXO1 binds to multiple crucial self-renewal genes and is required for their activation. In agreement with this observation, genetic and pharmacological ablation of FOXO1 inhibited the long-term proliferation and clonogenicity of AE cells and t(8;21) AML cell lines. Targeting of FOXO1 therefore provides a potential therapeutic strategy for elimination of stem cells at both preleukemic and leukemic stages.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteína Forkhead Box O1/metabolismo , Redes Reguladoras de Genes , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/metabolismo , Lesões Pré-Cancerosas/genética , Animais , Antígenos CD34/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Genoma Humano , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos SCID , Proteínas de Fusão Oncogênica/genética , Lesões Pré-Cancerosas/patologia , Proteína 1 Parceira de Translocação de RUNX1 , Regulação para Cima/genéticaRESUMO
Acute myeloid leukemia (AML) is a heterogeneous disease caused by mutations in transcriptional regulator genes, but how different mutant regulators shape the chromatin landscape is unclear. Here, we compared the transcriptional networks of two types of AML with chromosomal translocations of the RUNX1 locus that fuse the RUNX1 DNA-binding domain to different regulators, the t(8;21) expressing RUNX1-ETO and the t(3;21) expressing RUNX1-EVI1. Despite containing the same DNA-binding domain, the two fusion proteins display distinct binding patterns, show differences in gene expression and chromatin landscape, and are dependent on different transcription factors. RUNX1-EVI1 directs a stem cell-like transcriptional network reliant on GATA2, whereas that of RUNX1-ETO-expressing cells is more mature and depends on RUNX1. However, both types of AML are dependent on the continuous expression of the fusion proteins. Our data provide a molecular explanation for the differences in clinical prognosis for these types of AML.
Assuntos
Cromatina/metabolismo , Cromossomos Humanos/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Leucemia Mieloide Aguda/genética , Translocação Genética/genética , Sequência de Bases , Sítios de Ligação , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Sobrevivência Celular/genética , Fator de Transcrição GATA2/metabolismo , Técnicas de Silenciamento de Genes , Humanos , FenótipoRESUMO
The t(4;11)(q21;q23) fuses mixed-lineage leukemia (MLL) to AF4, the most common MLL-fusion partner. Here we show that MLL fused to murine Af4, highly conserved with human AF4, produces high-titer retrovirus permitting efficient transduction of human CD34+ cells, thereby generating a model of t(4;11) pro-B acute lymphoblastic leukemia (ALL) that fully recapitulates the immunophenotypic and molecular aspects of the disease. MLL-Af4 induces a B ALL distinct from MLL-AF9 through differential genomic target binding of the fusion proteins leading to specific gene expression patterns. MLL-Af4 cells can assume a myeloid state under environmental pressure but retain lymphoid-lineage potential. Such incongruity was also observed in t(4;11) patients in whom leukemia evaded CD19-directed therapy by undergoing myeloid-lineage switch. Our model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis.
Assuntos
Antígenos CD34/metabolismo , Transformação Celular Neoplásica/genética , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Translocação Genética , Animais , Linhagem da Célula , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismoRESUMO
Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect the epigenetic regulatory machinery and signaling molecules, leading to a block in hematopoietic differentiation. Constitutive signaling from mutated growth factor receptors is a major driver of leukemic growth, but how aberrant signaling affects the epigenome in AML is less understood. Furthermore, AML cells undergo extensive clonal evolution, and the mutations in signaling genes are often secondary events. To elucidate how chronic growth factor signaling alters the transcriptional network in AML, we performed a system-wide multi-omics study of primary cells from patients suffering from AML with internal tandem duplications in the FLT3 transmembrane domain (FLT3-ITD). This strategy revealed cooperation between the MAP kinase (MAPK) inducible transcription factor AP-1 and RUNX1 as a major driver of a common, FLT3-ITD-specific gene expression and chromatin signature, demonstrating a major impact of MAPK signaling pathways in shaping the epigenome of FLT3-ITD AML.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/enzimologia , Sistema de Sinalização das MAP Quinases , Mutação , Tirosina Quinase 3 Semelhante a fms/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Estrutura Terciária de Proteína , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Oncogenic transcription factors such as RUNX1/ETO, which is generated by the chromosomal translocation t(8;21), subvert normal blood cell development by impairing differentiation and driving malignant self-renewal. Here, we use digital footprinting and chromatin immunoprecipitation sequencing (ChIP-seq) to identify the core RUNX1/ETO-responsive transcriptional network of t(8;21) cells. We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion. Perturbation of this equilibrium in t(8;21) cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation. Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21) is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.
Assuntos
Leucemia Mieloide Aguda/patologia , Translocação Genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Redes Reguladoras de Genes , Humanos , Proteínas com Domínio LIM/metabolismo , Leucemia Mieloide Aguda/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Análise de Sequência de RNA , Transativadores/metabolismoAssuntos
Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteínas do Grupo Polycomb/genética , Receptores de Fatores de Crescimento/genética , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Mutação , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Translocação GenéticaRESUMO
The activation of B-cell-specific genes, such as CD19 and PAX5, is a hallmark of t(8;21) acute myeloid leukemia (AML) which expresses the translocation product RUNX1/ETO. PAX5 is an important regulator of B-lymphoid development and blocks myeloid differentiation when ectopically expressed. To understand the molecular mechanism of PAX5 deregulation, we examined its chromatin structure and regulation in t(8;21) AML cells, non-t(8;21) myeloid precursor control cells, and pre-B cells. In non-t(8;21) myeloid precursors, PAX5 is poised for transcription, but is repressed by polycomb complexes. In t(8;21) AML, PAX5 is not directly activated by RUNX1/ETO, but expression requires constitutive mitogen-activated protein (MAP) kinase signaling. Using a model of t(8;21) carrying an activating KIT mutation, we demonstrate that deregulated MAP kinase signaling in t(8;21) AML abrogates the association of polycomb complexes to PAX5 and leads to aberrant gene activation. Our findings therefore suggest a novel role of activating tyrosine kinase mutations in lineage-inappropriate gene expression in AML.