RESUMEN
Novel therapies for the treatment of acute myeloid leukemia (AML) are urgently needed, because current treatments do not cure most patients with AML. We report a domain-focused, kinome-wide CRISPR-Cas9 screening that identified protein kinase targets for the treatment of AML, which led to the identification of Rio-kinase 2 (RIOK2) as a potential novel target. Loss of RIOK2 led to a decrease in protein synthesis and to ribosomal instability followed by apoptosis in leukemic cells, but not in fibroblasts. Moreover, the ATPase function of RIOK2 was necessary for cell survival. When a small-molecule inhibitor was used, pharmacological inhibition of RIOK2 similarly led to loss of protein synthesis and apoptosis and affected leukemic cell growth in vivo. Our results provide proof of concept for targeting RIOK2 as a potential treatment of patients with AML.
Asunto(s)
Leucemia Mieloide Aguda , Inhibidores de Proteínas Quinasas , Animales , Ratones , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sistemas CRISPR-Cas , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Terapia Molecular Dirigida , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
KDM4/JMJD2 are H3K9- and H3K36-specific demethylases, which are considered promising therapeutic targets for the treatment of acute myeloid leukemia (AML) harboring MLL translocations. Here, we investigate the long-term effects of depleting KDM4 activity on normal hematopoiesis to probe potential side effects of continuous inhibition of these enzymes. Utilizing conditional Kdm4a/Kdm4b/Kdm4c triple-knockout mice, we show that KDM4 activity is required for hematopoietic stem cell (HSC) maintenance in vivo. The knockout of the KDM4 demethylases leads to accumulation of H3K9me3 on transcription start sites and the corresponding downregulation of expression of several genes in HSCs. We show that 2 of these genes, Taf1b and Nom1, are essential for the maintenance of hematopoietic cells. Taken together, our results show that the KDM4 demethylases are required for the expression of genes essential for the long-term maintenance of normal hematopoiesis.
Asunto(s)
Células Madre Hematopoyéticas/citología , Histona Demetilasas/genética , Animales , Supervivencia Celular , Células Cultivadas , Regulación de la Expresión Génica , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Sitio de Iniciación de la TranscripciónRESUMEN
Cardiac dysfunctions dramatically increase with age. Revealing a currently unknown contributor to cardiac ageing, we report the age-dependent, cardiac-specific accumulation of the lysosphingolipid sphinganine (dihydrosphingosine, DHS) as an evolutionarily conserved hallmark of the aged vertebrate heart. Mechanistically, the DHS-derivative sphinganine-1-phosphate (DHS1P) directly inhibits HDAC1, causing an aberrant elevation in histone acetylation and transcription levels, leading to DNA damage. Accordingly, the pharmacological interventions, preventing (i) the accumulation of DHS1P using SPHK2 inhibitors, (ii) the aberrant increase in histone acetylation using histone acetyltransferase (HAT) inhibitors, (iii) the DHS1P-dependent increase in transcription using an RNA polymerase II inhibitor, block DHS-induced DNA damage in human cardiomyocytes. Importantly, an increase in DHS levels in the hearts of healthy young adult mice leads to an impairment in cardiac functionality indicated by a significant reduction in left ventricular fractional shortening and ejection fraction, mimicking the functional deterioration of aged hearts. These molecular and functional defects can be partially prevented in vivo using HAT inhibitors. Together, we report an evolutionarily conserved mechanism by which increased DHS levels drive the decline in cardiac health.
Asunto(s)
Envejecimiento/genética , Envejecimiento/metabolismo , Variación Genética , Inestabilidad Genómica , Miocardio/metabolismo , Esfingolípidos/metabolismo , Animales , Curcumina/química , Curcumina/farmacología , Daño del ADN/efectos de los fármacos , Metabolismo Energético , Epigénesis Genética , Evolución Molecular , Fundulidae , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genómica/métodos , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Humanos , Modelos Moleculares , Miocitos Cardíacos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Relación Estructura-Actividad , Vertebrados/genética , Vertebrados/metabolismoRESUMEN
Regulation of protein synthesis is a key factor in hematopoietic stem cell maintenance and differentiation. Rio-kinase 2 (RIOK2) is a ribosome biogenesis factor that has recently been described an important regulator of human blood cell development. Additionally, we have previously identified RIOK2 as a regulator of protein synthesis and a potential target for the treatment of acute myeloid leukemia (AML). However, its functional relevance in several organ systems, including normal hematopoiesis, is not well understood. Here, we investigate the consequences of RIOK2 loss on normal hematopoiesis using two different conditional knockout mouse models. Using competitive and non-competitive bone marrow transplantations, we demonstrate that RIOK2 is essential for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the maintenance of fully differentiated blood cells in vivo as well as in vitro. Loss of RIOK2 leads to rapid death in full-body knockout mice as well as mice with RIOK2 loss specific to the hematopoietic system. Taken together, our results indicate that regulation of protein synthesis and ribosome biogenesis by RIOK2 is essential for the function of the hematopoietic system.
Asunto(s)
Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Animales , Humanos , Ratones , Trasplante de Médula Ósea , Diferenciación Celular/fisiología , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Ratones NoqueadosRESUMEN
Neurodegenerative disorders, such as Alzheimer's disease (AD) or Huntington's disease (HD), are linked to protein aggregate neurotoxicity. According to the "cholinergic hypothesis," loss of acetylcholine (ACh) signaling contributes to the AD pathology, and therapeutic restoration of ACh signaling is a common treatment strategy. How disease causation and the effect of ACh are linked to protein aggregation and neurotoxicity remains incompletely understood, thus limiting the development of more effective therapies. Here, we show that BAZ-2, the Caenorhabditis elegans ortholog of human BAZ2B, limits ACh signaling. baz-2 mutations reverse aggregation and toxicity of amyloid-beta as well as polyglutamine peptides, thereby restoring health and lifespan in nematode models of AD and HD, respectively. The neuroprotective effect of Δbaz-2 is mediated by choline acetyltransferase, phenocopied by ACh-esterase depletion, and dependent on ACh receptors. baz-2 reduction or ectopic ACh treatment augments proteostasis via induction of the endoplasmic reticulum unfolded protein response and the ubiquitin proteasome system.
Asunto(s)
Enfermedad de Alzheimer , Enfermedad de Huntington , Factores Generales de Transcripción , Animales , Humanos , Acetilcolina/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteínas que Contienen Bromodominio , Caenorhabditis elegans/metabolismo , Proteostasis , Factores Generales de Transcripción/metabolismoRESUMEN
Innate immune responses protect organisms against various insults, but may lead to tissue damage when aberrantly activated. In higher organisms, cytoplasmic DNA can trigger inflammatory responses that can lead to tissue degeneration. Simpler metazoan models could shed new mechanistic light on how inflammatory responses to cytoplasmic DNA lead to pathologies. Here, we show that in a DNase II-defective Caenorhabditis elegans strain, persistent cytoplasmic DNA leads to systemic tissue degeneration and loss of tissue functionality due to impaired proteostasis. These pathological outcomes can be therapeutically alleviated by restoring protein homeostasis, either via ectopic induction of the ER unfolded protein response or N-acetylglucosamine treatment. Our results establish C. elegans as an ancestral metazoan model for studying the outcomes of inflammation-like conditions caused by persistent cytoplasmic DNA and provide insight into potential therapies for human conditions involving chronic inflammation.
Asunto(s)
ADN/inmunología , Retículo Endoplásmico/metabolismo , Inmunidad Innata , Proteostasis , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismoRESUMEN
We present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.
RESUMEN
Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions.