RESUMEN
Chromosomal rearrangements of the mixed lineage leukemia (MLL) gene occur in â¼10% of B-cell acute lymphoblastic leukemia (B-ALL) and define a group of patients with dismal outcomes. Immunohistochemical staining of bone marrow biopsies from most of these patients revealed aberrant expression of BCL6, a transcription factor that promotes oncogenic B-cell transformation and drug resistance in B-ALL. Our genetic and ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analyses showed that MLL-AF4 and MLL-ENL fusions directly bound to the BCL6 promoter and up-regulated BCL6 expression. While oncogenic MLL fusions strongly induced aberrant BCL6 expression in B-ALL cells, germline MLL was required to up-regulate Bcl6 in response to physiological stimuli during normal B-cell development. Inducible expression of Bcl6 increased MLL mRNA levels, which was reversed by genetic deletion and pharmacological inhibition of Bcl6, suggesting a positive feedback loop between MLL and BCL6. Highlighting the central role of BCL6 in MLL-rearranged B-ALL, conditional deletion and pharmacological inhibition of BCL6 compromised leukemogenesis in transplant recipient mice and restored sensitivity to vincristine chemotherapy in MLL-rearranged B-ALL patient samples. Oncogenic MLL fusions strongly induced transcriptional activation of the proapoptotic BH3-only molecule BIM, while BCL6 was required to curb MLL-induced expression of BIM. Notably, peptide (RI-BPI) and small molecule (FX1) BCL6 inhibitors derepressed BIM and synergized with the BH3-mimetic ABT-199 in eradicating MLL-rearranged B-ALL cells. These findings uncover MLL-dependent transcriptional activation of BCL6 as a previously unrecognized requirement of malignant transformation by oncogenic MLL fusions and identified BCL6 as a novel target for the treatment of MLL-rearranged B-ALL.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Proteína de la Leucemia Mieloide-Linfoide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Animales , Biomarcadores de Tumor/genética , Supervivencia Celular/genética , Células Cultivadas , Eliminación de Gen , Marcación de Gen , Humanos , Ratones , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Pronóstico , Regiones Promotoras Genéticas/genéticaRESUMEN
BACKGROUND: The burden of COVID-19 disease can be measured in terms of disability-adjusted life years (DALYs), which is composed of two components: the years of life lost through premature death (YLL) and the number of years lived with disability (YLD), adjusted for level of disability. This study measured DALYs due to COVID-19 in Sweden and compared it to the burden of other diseases. METHODS: The methodology used in the calculation of DALYs was based on the Global Burden of Disease guidelines. The number of patients diagnosed with mild/moderate, severe or critical COVID-19 and/or post-COVID-19 condition between March 2020 and October 2021 was extracted from national registries and used for YLD calculations. In addition, the numbers of death due to COVID-19 in different age groups were used for the YLL calculation. RESULTS: During the study period, 152,877 DALYs were lost to COVID-19 in Sweden, 99.3% of which was attributed to YLL. Loss of DALYs occurred mainly among the elderly, with 66.8% of DALYs attributed to individuals >70 years old. Compared to other diseases, the burden of COVID-19 in 2020 ranked as the eighth leading cause of DALY lost. CONCLUSIONS: Similar to other countries, the burden of COVID-19 in Sweden was concentrated mainly among the elderly, who contributed most of the DALY lost due to premature mortality. Yet, DALY loss remained lower for COVID-19 than for several other diseases. The contribution of YLD to DALYs lost was minimal. However empirical data on the occurrence and disability of post-COVID-19 condition are scarce, and YLD may therefore be underestimated.
Asunto(s)
COVID-19 , Personas con Discapacidad , Humanos , Anciano , Años de Vida Ajustados por Discapacidad , Años de Vida Ajustados por Calidad de Vida , Suecia/epidemiología , COVID-19/epidemiología , Costo de EnfermedadRESUMEN
B lymphocyte differentiation is an exquisitely regulated homeostatic process resulting in continuous production of appropriately selected B cells. Relatively small changes in gene expression can result in deregulation of this process, leading to acute lymphocytic leukemia (ALL), immune deficiency, or autoimmunity. Translocation of MLL1 (KMT2A) often results in a pro-B cell ALL, but little is known about its role in normal B cell differentiation. Using a Rag1-cre mouse knock-in to selectively delete Mll1 in developing lymphocytes, we show that B cell, but not T cell, homeostasis depends on MLL1. Mll1-/- B progenitors fail to differentiate efficiently through the pro- to pre-B cell transition, resulting in a persistent reduction in B cell populations. Cells inefficiently transit the pre-BCR checkpoint, despite normal to higher levels of pre-BCR components, and rearranged IgH expression fails to rescue this differentiation block. Instead of IgH-rearrangement defects, we find that Mll1-/- pre-B cells exhibit attenuated RAS/MAPK signaling downstream of the pre-BCR, which results in reduced survival in physiologic levels of IL-7. Genome-wide expression data illustrate that MLL1 is connected to B cell differentiation and IL-7-dependent survival through a complex transcriptional network. Overall, our data demonstrate that wild-type MLL1 is a regulator of pre-BCR signaling and B cell differentiation and further suggest that targeting its function in pro-B cell ALL may be more broadly effective than previously anticipated.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , N-Metiltransferasa de Histona-Lisina/metabolismo , Interleucina-7/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Animales , Células Cultivadas , Femenino , Homeostasis/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Transducción de Señal/inmunologíaRESUMEN
PURPOSE OF REVIEW: Histone H3, lysine 4 (H3K4) methylation is one chromatin modification that defines distinct regulatory states of euchromatin. Mammals express six main histone methyltransferase (HMT) enzymes that modify H3K4 by monomethylation, dimethylation or trimethylation. Recent studies examine roles of some of these HMTs and their cofactors in hematopoiesis and leukemia. We discuss these emerging studies together with prior embryonic stem data, revealing how these enzymes function. RECENT FINDINGS: Murine models have been employed to conditionally or constitutively knockout HMTs (MLL1/KMT2A, MLL2/KMT2B, MLL3/KMT2C, MLL4/KMT2D, SETD1A/KMT2F and SETD1B/KMT2G) as well as specific domains or partners of these enzymes in normal hematopoietic populations and in the context of hematologic malignancies. These studies demonstrate that global or gene-specific changes in H3K4 modification levels can be attributed to particular enzymes in particular tissues. SUMMARY: Loss-of-function studies indicate largely nonoverlapping roles of the six H3K4 HMTs. These roles are not all necessarily due to differences in enzymatic activity and are not always accompanied by large global changes in histone modification. Both gain-of-function and loss-of-function mutations in hematologic malignancy are restricted to MLL1 and MLL3/MLL4, but emerging data indicate that SETD1A/SETD1B and MLL2 can be critical in leukemia as well.
Asunto(s)
Neoplasias Hematológicas/metabolismo , Hematopoyesis , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Animales , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Humanos , Metilación , Familia de Multigenes , Especificidad de Órganos , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Neuronal histone H3-lysine 4 methylation landscapes are defined by sharp peaks at gene promoters and other cis-regulatory sequences, but molecular and cellular phenotypes after neuron-specific deletion of H3K4 methyl-regulators remain largely unexplored. We report that neuronal ablation of the H3K4-specific methyltransferase, Kmt2a/Mixed-lineage leukemia 1 (Mll1), in mouse postnatal forebrain and adult prefrontal cortex (PFC) is associated with increased anxiety and robust cognitive deficits without locomotor dysfunction. In contrast, only mild behavioral phenotypes were observed after ablation of the Mll1 ortholog Kmt2b/Mll2 in PFC. Impaired working memory after Kmt2a/Mll1 ablation in PFC neurons was associated with loss of training-induced transient waves of Arc immediate early gene expression critical for synaptic plasticity. Medial prefrontal layer V pyramidal neurons, a major output relay of the cortex, demonstrated severely impaired synaptic facilitation and temporal summation, two forms of short-term plasticity essential for working memory. Chromatin immunoprecipitation followed by deep sequencing in Mll1-deficient cortical neurons revealed downregulated expression and loss of the transcriptional mark, trimethyl-H3K4, at <50 loci, including the homeodomain transcription factor Meis2. Small RNA-mediated Meis2 knockdown in PFC was associated with working memory defects similar to those elicited by Mll1 deletion. Therefore, mature prefrontal neurons critically depend on maintenance of Mll1-regulated H3K4 methylation at a subset of genes with an essential role in cognition and emotion.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Memoria a Corto Plazo/fisiología , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Plasticidad Neuronal/fisiología , Corteza Prefrontal/fisiología , Animales , Conducta Animal/fisiología , Proteínas del Citoesqueleto/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/efectos de los fármacos , Proteínas de Homeodominio/genética , Masculino , Metilación , Ratones , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Prosencéfalo/fisiología , Células Piramidales/fisiologíaRESUMEN
The histone methyltransferase Mixed Lineage Leukemia (MLL) is essential to maintain hematopoietic stem cells and is a leukemia protooncogene. Although clustered homeobox genes are well-characterized targets of MLL and MLL fusion oncoproteins, the range of Mll-regulated genes in normal hematopoietic cells remains unknown. Here, we identify and characterize part of the Mll-dependent transcriptional network in hematopoietic stem cells with an integrated approach by using conditional loss-of-function models, genomewide expression analyses, chromatin immunoprecipitation, and functional rescue assays. The Mll-dependent transcriptional network extends well beyond the previously appreciated Hox targets, is comprised of many characterized regulators of self-renewal, and contains target genes that are both dependent and independent of the MLL cofactor, Menin. Interestingly, PR-domain containing 16 emerged as a target gene that is uniquely effective at partially rescuing Mll-deficient hematopoietic stem and progenitor cells. This work highlights the tissue-specific nature of regulatory networks under the control of MLL/Trithorax family members and provides insight into the distinctions between the participation of MLL in normal hematopoiesis and in leukemia.
Asunto(s)
Epigénesis Genética/fisiología , Redes Reguladoras de Genes/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/fisiología , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Inmunoprecipitación de Cromatina , N-Metiltransferasa de Histona-Lisina , Humanos , Modelos Biológicos , Proteínas Proto-Oncogénicas/fisiologíaRESUMEN
Mixed Lineage Leukemia (MLL1) translocations encode fusion proteins retaining the N terminus of MLL1, which interacts with the tumor suppressor, menin. This interaction is essential for leukemogenesis and thus is a promising drug target. However, wild-type MLL1 plays a critical role in sustaining hematopoietic stem cells (HSCs); therefore, disruption of an essential MLL1 cofactor would be expected to obliterate normal hematopoiesis. Here we show that rather than working together as a complex, menin and MLL1 regulate distinct pathways during normal hematopoiesis, particularly in HSCs and B cells. We demonstrate the lack of genetic interaction between menin and MLL1 in steady-state or regenerative hematopoiesis and in B-cell differentiation despite the fact that MLL1 is critical for these processes. In B cells, menin- or MLL1-regulated genes can be classified into 3 categories: (1) a relatively small group of coregulated genes including previously described targets Hoxa9 and Meis1 but also Mecom and Eya1, and much larger groups of (2) exclusively menin-regulated and (3) exclusively MLL1-regulated genes. Our results highlight the large degree of independence of these 2 proteins and demonstrate that menin is not a requisite cofactor for MLL1 during normal hematopoiesis. Furthermore, our data support the development of menin-MLL1-disrupting drugs as safe and selective leukemia targeting agents.
Asunto(s)
Linfocitos B/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Animales , Linfocitos B/citología , Diferenciación Celular/fisiología , Epistasis Genética , Femenino , Regulación de la Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Linfopoyesis/genética , Ratones , Ratones Transgénicos , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Epigenetic mechanisms that maintain neurogenesis throughout adult life remain poorly understood. Trithorax group (trxG) and Polycomb group (PcG) gene products are part of an evolutionarily conserved chromatin remodelling system that activate or silence gene expression, respectively. Although PcG member Bmi1 has been shown to be required for postnatal neural stem cell self-renewal, the role of trxG genes remains unknown. Here we show that the trxG member Mll1 (mixed-lineage leukaemia 1) is required for neurogenesis in the mouse postnatal brain. Mll1-deficient subventricular zone neural stem cells survive, proliferate and efficiently differentiate into glial lineages; however, neuronal differentiation is severely impaired. In Mll1-deficient cells, early proneural Mash1 (also known as Ascl1) and gliogenic Olig2 expression are preserved, but Dlx2, a key downstream regulator of subventricular zone neurogenesis, is not expressed. Overexpression of Dlx2 can rescue neurogenesis in Mll1-deficient cells. Chromatin immunoprecipitation demonstrates that Dlx2 is a direct target of MLL in subventricular zone cells. In differentiating wild-type subventricular zone cells, Mash1, Olig2 and Dlx2 loci have high levels of histone 3 trimethylated at lysine 4 (H3K4me3), consistent with their transcription. In contrast, in Mll1-deficient subventricular zone cells, chromatin at Dlx2 is bivalently marked by both H3K4me3 and histone 3 trimethylated at lysine 27 (H3K27me3), and the Dlx2 gene fails to properly activate. These data support a model in which Mll1 is required to resolve key silenced bivalent loci in postnatal neural precursors to the actively transcribed state for the induction of neurogenesis, but not for gliogenesis.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Neurogénesis , Neuronas/citología , Células Madre/citología , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Metilación , Ratones , Proteína de la Leucemia Mieloide-Linfoide/deficiencia , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/metabolismo , Bulbo Olfatorio/citología , Bulbo Olfatorio/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos , Células Madre/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: To investigate the role of acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in hematopoiesis. APPROACH AND RESULTS: ACAT1 converts cellular cholesterol to cholesteryl esters for storage in multiple cell types and is a potential drug target for human diseases. In mouse models for atherosclerosis, global Acat1 knockout causes increased lesion size; bone marrow transplantation experiments suggest that the increased lesion size might be caused by ACAT1 deficiency in macrophages. However, bone marrow contains hematopoietic stem cells that give rise to cells in myeloid and lymphoid lineages; these cell types affect atherosclerosis at various stages. Here, we test the hypothesis that global Acat1(-/-) may affect hematopoiesis, rather than affecting macrophage function only, and show that Acat1(-/-) mice contain significantly higher numbers of myeloid cells and other cells than wild-type mice. Detailed analysis of bone marrow cells demonstrated that Acat1(-/-) causes a higher proportion of the stem cell-enriched Lin(-)Sca-1(+)c-Kit(+) population to proliferate, resulting in higher numbers of myeloid progenitor cells. In addition, we show that Acat1(-/-) causes higher monocytosis in Apoe(-/-) mouse during atherosclerosis development. CONCLUSIONS: ACAT1 plays important roles in hematopoiesis in normal mouse and in Apoe(-/-) mouse during atherosclerosis development.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/deficiencia , Aterosclerosis/enzimología , Células de la Médula Ósea/enzimología , Proliferación Celular , Hematopoyesis , Células Madre Hematopoyéticas/enzimología , Leucocitosis/enzimología , Acetil-CoA C-Acetiltransferasa/genética , Animales , Antígenos Ly/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/genética , Aterosclerosis/inmunología , Biomarcadores/metabolismo , Linaje de la Célula , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Genotipo , Leucocitosis/genética , Leucocitosis/inmunología , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Células Precursoras de Linfocitos B/enzimología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factores de TiempoRESUMEN
Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR-Cas9 screens, we identify specific roles for MTF2-PRC2.1, PCGF1-PRC1.1 and Menin-KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin-KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.
Asunto(s)
Células Madre Pluripotentes , Factores de Transcripción , Proteínas del Grupo Polycomb/genética , Factores de Transcripción/genética , Genoma , Regiones Promotoras GenéticasRESUMEN
Human pluripotent stem cells, including embryonic (hES) and induced pluripotent stem cells (hiPS), retain the ability to self-renew indefinitely, while maintaining the capacity to differentiate into all cell types of the nervous system. While human pluripotent cell-based therapies are unlikely to arise soon, these cells can currently be used as an inexhaustible source of committed neurons to perform high-throughput screening and safety testing of new candidate drugs. Here, we describe critically the available methods and molecular factors that are used to direct the differentiation of hES or hiPS into specific neurons. In addition, we discuss how the availability of patient-specific hiPS offers a unique opportunity to model inheritable neurodegenerative diseases and untangle their pathological mechanisms, or to validate drugs that would prevent the onset or the progression of these neurological disorders.
Asunto(s)
Células Madre Embrionarias/citología , Enfermedades Neurodegenerativas/metabolismo , Células Madre Pluripotentes/citología , Animales , Diferenciación Celular , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/patología , Humanos , Enfermedades Neurodegenerativas/patología , Neuronas/citología , Neuronas/metabolismo , Neuronas/patología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patologíaRESUMEN
Abnormal activation of SETBP1 due to overexpression or missense mutations occurs frequently in various myeloid neoplasms and associates with poor prognosis. Direct activation of Hoxa9/Hoxa10/Myb transcription by SETBP1 and its missense mutants is essential for their transforming capability; however, the underlying epigenetic mechanisms remain elusive. We found that both SETBP1 and its missense mutant SETBP1(D/N) directly interact with histone methyltransferase MLL1. Using a combination of ChIP-seq and RNA-seq analysis in primary hematopoietic stem and progenitor cells, we uncovered extensive overlap in their genomic occupancy and their cooperation in activating many oncogenic transcription factor genes including Hoxa9/Hoxa10/Myb and a large group of ribosomal protein genes. Genetic ablation of Mll1 as well as treatment with an inhibitor of the MLL1 complex OICR-9429 abrogated Setbp1/Setbp1(D/N)-induced transcriptional activation and transformation. Thus, the MLL1 complex plays a critical role in Setbp1-induced transcriptional activation and transformation and represents a promising target for treating myeloid neoplasms with SETBP1 activation.
RESUMEN
The clinical success of engineered, CD19-directed chimeric antigen receptor (CAR) T cells in relapsed, refractory B-cell acute lymphoblastic leukemia (B-ALL) has generated great enthusiasm for the use of CAR T cells in patients with cytogenetics that portend a poor prognosis with conventional cytotoxic therapies. One such group includes infants and children with mixed lineage leukemia (MLL1, KMT2A) rearrangements (MLL-r), who fare much worse than patients with low- or standard-risk B-ALL. Although early clinical trials using CD19 CAR T cells for MLL-r B-ALL produced complete remission in most patients, relapse with CD19-negative disease was a common mechanism of treatment failure. Whereas CD19neg relapse has been observed across a broad spectrum of B-ALL patients treated with CD19-directed therapy, patients with MLL-r have manifested the emergence of AML, often clonally related to the B-ALL, suggesting that the inherent heterogeneity or lineage plasticity of MLL-r B-ALL may predispose patients to a myeloid relapse. Understanding the factors that enable and drive myeloid relapse may be important to devise strategies to improve durability of remissions. In this review, we summarize clinical observations to date with MLL-r B-ALL and generally discuss lineage plasticity as a mechanism of escape from immunotherapy.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/genética , Inmunoterapia Adoptiva , Proteína de la Leucemia Mieloide-Linfoide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Animales , Antígenos CD19/genética , Antígenos CD19/inmunología , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina/inmunología , Humanos , Inmunoterapia Adoptiva/métodos , Proteína de la Leucemia Mieloide-Linfoide/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Resultado del Tratamiento , Escape del TumorRESUMEN
Translocations of Meningioma-1 (MN1) occur in a subset of acute myeloid leukemias (AML) and result in high expression of MN1, either as a full-length protein, or as a fusion protein that includes most of the N-terminus of MN1. High levels of MN1 correlate with poor prognosis. When overexpressed in murine hematopoietic progenitors, MN1 causes an aggressive AML characterized by an aberrant myeloid precursor-like gene expression program that shares features of KMT2A-rearranged (KMT2A-r) leukemia, including high levels of Hoxa and Meis1 gene expression. Compounds that target a critical KMT2A-Menin interaction have proven effective in KMT2A-r leukemia. Here, we demonstrate that Menin (Men1) is also critical for the self-renewal of MN1-driven AML through the maintenance of a distinct gene expression program. Genetic inactivation of Men1 led to a decrease in the number of functional leukemia-initiating cells. Pharmacologic inhibition of the KMT2A-Menin interaction decreased colony-forming activity, induced differentiation programs in MN1-driven murine leukemia and decreased leukemic burden in a human AML xenograft carrying an MN1-ETV6 translocation. Collectively, these results nominate Menin inhibition as a promising therapeutic strategy in MN1-driven leukemia.
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Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Proteínas Supresoras de Tumor/genética , Animales , Línea Celular Tumoral , Regulación Leucémica de la Expresión Génica/genética , Células HEK293 , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Ratones , Ratones NoqueadosRESUMEN
ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.
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Antineoplásicos/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Leucemia/tratamiento farmacológico , Leucemia/enzimología , Animales , Antineoplásicos/química , Dominio Catalítico/efectos de los fármacos , Dominio Catalítico/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Diseño de Fármacos , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Femenino , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Leucemia/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Proteína de la Leucemia Mieloide-Linfoide/genética , Oncogenes , Dominios Proteicos , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Organogenesis is dependent on the formation of distinct cell types within the embryo. Important to this process are the hox genes, which are believed to confer positional identities to cells along the anteroposterior axis. Here, we have identified the caudal-related gene cdx4 as the locus mutated in kugelig (kgg), a zebrafish mutant with an early defect in haematopoiesis that is associated with abnormal anteroposterior patterning and aberrant hox gene expression. The blood deficiency in kgg embryos can be rescued by overexpressing hoxb7a or hoxa9a but not hoxb8a, indicating that the haematopoietic defect results from perturbations in specific hox genes. Furthermore, the haematopoietic defect in kgg mutants is not rescued by scl overexpression, suggesting that cdx4 and hox genes act to make the posterior mesoderm competent for blood development. Overexpression of cdx4 during zebrafish development or in mouse embryonic stem cells induces blood formation and alters hox gene expression. Taken together, these findings demonstrate that cdx4 regulates hox genes and is necessary for the specification of haematopoietic cell fate during vertebrate embryogenesis.
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Genes Homeobox/fisiología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/metabolismo , Mutación/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Tipificación del Cuerpo , Línea Celular , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/genética , Genotipo , Hematopoyesis , Células Madre Hematopoyéticas/citología , Proteínas de Homeodominio/genética , Riñón/embriología , Ratones , Datos de Secuencia Molecular , Familia de Multigenes/genética , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The Mixed Lineage Leukemia (MLL1, KMT2A) gene is critical for development and maintenance of hematopoietic stem cells (HSCs), however, whether this protein is limiting for HSC development is unknown due to lack of physiologic model systems. Here, we develop an MLL1-inducible embryonic stem cell (ESC) system and show that induction of wild-type MLL1 during ESC differentiation selectively increases hematopoietic potential from a transitional c-Kit+/Cd41+ population in the embryoid body and also at sites of hematopoiesis in embryos. Single-cell sequencing analysis illustrates inherent heterogeneity of the c-Kit+/Cd41+ population and demonstrates that MLL1 induction shifts its composition toward multilineage hematopoietic identities. Surprisingly, this does not occur through increasing Hox or other canonical MLL1 targets but through an enhanced Rac/Rho/integrin signaling state, which increases responsiveness to Vla4 ligands and enhances hematopoietic commitment. Together, our data implicate a Rac/Rho/integrin signaling axis in the endothelial to hematopoietic transition and demonstrate that MLL1 actives this axis.
Asunto(s)
Hematopoyesis , N-Metiltransferasa de Histona-Lisina/metabolismo , Integrinas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Biomarcadores/metabolismo , Adhesión Celular , Diferenciación Celular , Ensayo de Unidades Formadoras de Colonias , Cuerpos Embrioides/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Mesodermo/citología , RatonesRESUMEN
The Mixed-Lineage Leukemia (MLL) gene encodes a Trithorax-related chromatin-modifying protooncogene that positively regulates Hox genes. In addition to their well-characterized roles in axial patterning, Trithorax and Polycomb family proteins perform less-understood functions in vertebrate hematopoiesis. To define the role of MLL in the development of the hematopoietic system, we examined the potential of cells lacking MLL. Mll-deficient cells could not develop into lymphocytes in adult RAG-2 chimeric animals. Similarly, in vitro differentiation of B cells required MLL. In chimeric embryos, Mll-deficient cells failed to contribute to fetal liver hematopoietic stem cell/progenitor populations. Moreover, we show that aorta-gonad-mesonephros (AGM) cells from Mll-deficient embryos lacked hematopoietic stem cell (HSC) activity despite their ability to generate hematopoietic progeny in vitro. These results demonstrate an intrinsic requirement for MLL in definitive hematopoiesis, where it is essential for the generation of HSCs in the embryo.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/fisiología , Proto-Oncogenes , Células Madre/fisiología , Animales , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Aorta/citología , Aorta/embriología , Aorta/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo/métodos , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Citometría de Flujo/métodos , Galactósidos/metabolismo , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas , N-Metiltransferasa de Histona-Lisina , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Indoles/metabolismo , Células Asesinas Naturales/fisiología , Antígenos Comunes de Leucocito/metabolismo , Hígado/citología , Hígado/metabolismo , Linfopoyesis/efectos de los fármacos , Linfopoyesis/fisiología , Mesonefro/citología , Mesonefro/embriología , Mesonefro/metabolismo , Ratones , Ratones Transgénicos , Proteína de la Leucemia Mieloide-Linfoide , Fenotipo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Trasplante de Células Madre/métodos , Células Madre/citología , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The deregulation of hematopoietic stem cell (HSC) transcriptional networks is a common theme in acute myelogenous leukemia (AML). Chromosomal translocations that alter the Mixed Lineage Leukemia 1 gene (MLL1, MLL, KMT2A) occur in infant, childhood and adult leukemia and at the same time, wild-type MLL1 is a critical regulator of HSC homeostasis. Typically, the endogenous, wild-type (WT) MLL1 and MLL fusion oncoproteins (MLL-FPs) remain both expressed in leukemia. WT and MLL-FPs activate overlapping sets of target genes, presenting a challenge for the selective therapeutic targeting of leukemic cells. We previously demonstrated that endogenous MLL1 is not required for the maintenance of MLL-FP-driven AML but is required for normal HSC homeostasis. Here we address the role of MLL-FPs in the initiation of leukemia in the absence of endogenous MLL1. We show that loss of endogenous Mll1 results in a rapid decrease in expression of shared HSC/leukemia target genes, yet MLL-AF9 restores the expression of most of these target genes in the absence of WT MLL1, with the critical exception of Mecom/Evi1. These observations underscore the sufficiency of MLL-fusion oncoproteins for initiating leukemia, but also illustrate that WT MLL1 target genes differ in their ability to be re-activated by MLL-FPs.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Animales , Antineoplásicos/farmacología , Carcinogénesis/genética , Carcinogénesis/metabolismo , Femenino , Regulación Leucémica de la Expresión Génica , Células HEK293 , Trasplante de Células Madre Hematopoyéticas , Humanos , Leucemia Mieloide Aguda/metabolismo , Proteína del Locus del Complejo MDS1 y EV11/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Fusión Oncogénica/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologíaRESUMEN
Disrupting the protein-protein interaction for molecularly targeted cancer therapeutics can be a challenging but promising strategy. Compounds that disrupt the interaction between menin, a chromatin-binding protein, and oncogenic mixed lineage leukemia fusion proteins (MLL-FPs) have shown significant promise in preclinical models of leukemia and have a high degree of selectivity for leukemia versus normal hematopoietic cells. Biochemical and structural studies demonstrate that, in addition to disrupting the menin-MLL-FP interaction, such compounds also inhibit menin-MLL1, menin-MLL2, and other menin-interacting proteins. Here, we address the degree to which disruption of menin-MLL-FP interactions or menin-MLL1/MLL2 interactions contribute to the antileukemia effect of menin inhibition. We show that Men1 deletion in MLL-AF9-transformed leukemia cells produces distinct cellular and molecular consequences compared with Mll1;Mll2 co-deletion and that compounds disrupting menin-MLL N-terminal interactions largely phenocopy menin loss. Moreover, we show that Mll1;Mll2-deficient leukemia cells exhibit enhanced sensitivity to menin interaction inhibitors, which is consistent with each regulating complementary genetic pathways. These data illustrate the heightened dependency of MLL-FPs on menin compared with wild-type MLL1/MLL2 for regulation of downstream target genes and argue that the predominant action of menin inhibitory compounds is through direct inhibition of MLL-FPs without significant contribution from MLL1/MLL2 inhibition.