Untersuchungen zur Regulierung und Funktion der Mitogen-aktivierten Proteinkinase ERK5 = Regulation and Function of the Mitogen-activated Protein Kinase ERK5

Mitogen activated protein kinases (MAPKs) are found in all eukaryotic cells and represent crucial elements in the signal transduction from the plasma membrane to the nucleus. Although a broad variety of extracellular stimuli activate MAPKs, they evoke very distinct cellular responses. The amplitude and duration of MAPK activation determine signal identity and ultimately cell fate. A tight and finely tuned regulation is therefore critical for a specific cellular response. The role and the regulation of extracellular signal-regulated kinase 5 (ERK5), a MAPK with a large and unique C-terminal tail, were studied in different cellular systems. The study highlights two aspects of ERK5 regulation: control of the phosphorylation state and regulated protein stability. In analogy to other MAPKs ERK5 is activated by dual phosphorylation of threonine and tyrosine residues in its activation motif. A first part of the study concentrates on whether and how the protein tyrosine phosphatase PTP-SL is involved in the downregulation of the ERK5 signal. The direct interaction of both proteins is shown to result in mutual modulation of their enzymatic activities. PTP-SL is a substrate of ERK5 and, independent of its phosphorylation, binding to the kinase enhances its catalytic phosphatase activity. On the other hand, interaction with PTP-SL does not only downregulate enzymatic ERK5 activity but also effectively impedes its translocation to the nucleus. The second part of this study focuses on the interaction of ERK5 with c-Abl and its oncogenic variants Bcr/Abl and v-Abl. In this study these tyrosine kinases are demonstrated to regulate ERK5 by two mechanisms: first, by induction of kinase activity and secondly, by stabilisation of the ERK5 protein. Stabilisation involves the direct interaction of unique ERK5 domains with Abl kinases and is independent of MAPK cascade activation. The level of ERK5 and its intrinsic basal activity – rather than its activation – are essential for v-Abl-induced transformation as well as for survival of Bcr/Abl-positive leukaemia cells. Stabilisation of ERK5 thus contributes to cell survival and should therefore be considered as an additional aspect in therapy of chronic myeloid leukaemia. Taken together, the results obtained in this study demonstrate that diverse pathways regulate ERK5 signalling by affecting kinase activity, localisation and protein stability. While the phosphatase PTP-SL is involved in negative regulation of ERK5, Abl kinases potently activate ERK5 and increase its half-life. Protein stabilisation thus is presented as a novel mechanism in the regulation of MAPKs.Mitogen-aktivierte Proteinkinasen (MAPKn) werden in allen eukaryontischen Zellen exprimiert und spielen eine bedeutende Rolle in der Weiterleitung von Signalen von der Plasmamembran zum Zellkern. Obwohl eine Vielzahl von unterschiedlichsten Stimulanzien MAPKn aktiveren, rufen diese doch sehr spezifische und vor allem adequate Reaktionen der Zelle hervor. Die molekularen Grundlagen für dieses Pradoxon sind weitgehend unbekannt. Es ist allerdings klar, daß die Amplitude und die Dauer der MAPKn-Aktivierung zwei entscheidende Parameter sind, die den weiteren Signalweg definieren und somit das Schicksal der Zelle festlegen. Daher müssen MAPKn einer sehr stringenten und fein regulierbaren Kontrolle unterliegen. In der vorliegenden Arbeit wurde die Funktion und die Regulierung der durch extrazelluläre Signale regulierten Kinase 5 (ERK5), einer MAPK mit einem außergewöhnlich langen und strukturell einzigartigen C-Terminus, in unterschiedlichen Zellsystemen untersucht. Zwei Aspekte der Regulierung von ERK5 werden insbesonders hervorgehoben: die Kontrolle des Phosphorilierungsstatus und die beeinflußbare Proteinstabilität. In Analogie zu anderen MAPKs wird ERK5 durch die Phosphorilierung eines Threonin und eines Tyrosinrestes aktiviert. Der erste Teil dieser Studie konzentriert sich auf die Frage ob und wie die Phosphatase PTP-SL und der Regulierung der ERK5 beteiligt sein könnte. Es konnte gezeigt werden, daß die direkte Interaktion dieser beiden Proteine zur gegenseitigen Beeinflussung ihrer enzymatischen Aktivitäten führt. PTP-SL ist nicht nur ein Substrat für die ERK5 sondern besitzt auch im Komplex mit ERK5 eine höhere Aktivität. Andererseits ist PTP-SL in der Lage, die Aktivität von ERK5 runterzuregulieren und darüber hinaus auch die Translokation von ERK5 in den Nucleus zu inhibieren. Der zweite Scherpunkt dieser Arbeit liegt auf der Wechselwirkung von ERK5 mit der Tyrosinkinasen c-Abl und ihren onkogenen Varianten Bcr/Abl und v-Abl. Es konnte gezeigt werden, daß diese Tyrosinkinasen ERK5 in zweierlei Weise beeinflussen: Erstens führen sie zur klassischen Aktivierung von ERK5 und zweitens stabilisieren sie das ERK5-Protein. Die Bedeutung der Proteinstabilisierung wurde durch die Untersuchung der Funktion von ERK5 bei von Abl-Kinase vermittelten Prozessen veranschaulicht. Sowohl für die Vestärkung Transformierung von Nagerfibroblasten durch v-Abl als auch für das Überleben von Bcr/Abl-positiven Leukämiezellen waren die Proteinmenge und die Basalaktivität und nicht etwa die Aktivierung von ERK5 ausschlaggebend. Zusammengenommen zeigen die Ergebnisse dieser Arbeit, daß die ERK5-vermittelte Signale durch Beeinflussung der Kinaseaktivität, der zellulären Lokalisation und der Proteinstabilität von ERK5 reguliert werden können. Während die Phosphatase PTP-SL an der Negativregulierung der ERK5 beteiligt ist, verstärken Abl-Kinasen ihre Aktivität und verlängern darüberhinaus die Halbwertszeit des Proteines in der Zelle. Proteinstabilisierung stellt somit einen neuen Aspekt in der Kontrolle von MAPKn dar

Elongator: An Ancestral Complex Driving Transcription and Migration through Protein Acetylation

Elongator is an evolutionary highly conserved complex. At least two of its cellular functions rely on the intrinsic lysine acetyl-transferase activity of the Elongator complex. Its two known substrates—Histone H3 and α-Tubulin—reflect the different roles of Elongator in the cytosol and the nucleus. A picture seems to emerge in which nuclear Elongator could regulate the transcriptional elongation of a subset of stress-inducible genes through acetylation of Histone H3 in the promoter-distal gene body. In the cytosol, Elongator-mediated acetylation of α-Tubulin contributes to intracellular trafficking and cell migration. Defects in both functions of Elongator have been implicated in neurodegenerative disorders

Post-Translational Modifications of H2A Histone Variants and Their Role in Cancer

Altres ajuts: Research leading to inventions at the IJC is supported by the 'La Caixa' Foundation, the Fundació Internacional Josep Carreras, Celgene Spain and the CERCA Programme/Generalitat de Catalunya.Histone variants are chromatin components that replace replication-coupled histones in a fraction of nucleosomes and confer particular characteristics to chromatin. H2A variants represent the most numerous and diverse group among histone protein families. In the nucleosomal structure, H2A-H2B dimers can be removed and exchanged more easily than the stable H3-H4 core. The unstructured N-terminal histone tails of all histones, but also the C-terminal tails of H2A histones protrude out of the compact structure of the nucleosome core. These accessible tails are the preferential target sites for a large number of post-translational modifications (PTMs). While some PTMs are shared between replication-coupled H2A and H2A variants, many modifications are limited to a specific histone variant. The present review focuses on the H2A variants H2A.Z, H2A.X, and macroH2A, and summarizes their functions in chromatin and how these are linked to cancer development and progression. H2A.Z primarily acts as an oncogene and macroH2A and H2A.X as tumour suppressors. We further focus on the regulation by PTMs, which helps to understand a degree of context dependency

Epigenetics in a spectrum of myeloid diseases and its exploitation for therapy

Mutations in genes encoding chromatin regulators are early events contributing to de-veloping asymptomatic clonal hematopoiesis of indeterminate potential and its frequent progression to myeloid diseases with increasing severity. We focus on the subset of myeloid diseases encompassing myelodysplastic syndromes and their transformation to secondary acute myeloid leukemia. We introduce the major concepts of chromatin regulation that provide the basis of epigenetic regulation. In greater detail, we discuss those chromatin regulators that are frequently mutated in myelodysplastic syndromes. We discuss their role in the epigenetic regulation of normal hematopoiesis and the consequence of their mutation. Finally, we provide an update on the drugs interfering with chromatin regulation approved or in development for myelodysplastic syndromes and acute myeloid leukemia

The MacroH2A1.1 – PARP1 Axis at the Intersection Between Stress Response and Metabolism

The exchange of replication-coupled canonical histones by histone variants endows chromatin with specific features. The replacement of the canonical H2A histone for the histone variant macroH2A is one of the most remarkable epigenetic modifications. The three vertebrate macroH2A proteins have a unique tripartite structure consisting of H2A-like domain, unstructured linker, and macrodomain. Macrodomains are ancient globular folds that are able to bind nicotinamide adenine dinucleotide (NAD+) derived metabolites. Here, we will briefly describe the physiological relevance of the metabolite binding in the context of chromatin. In particular, we will focus on the macroH2A1.1 isoform that binds ADP-ribose and poly-ADP-ribose polymerase 1 (PARP1) enzyme, a cellular stress sensor. We will discuss the impact of this interaction in the context of cancer, senescence, cell stress and energy metabolism

A clinical-molecular update on azanucleoside-based therapy for the treatment of hematologic cancers

The azanucleosides azacitidine and decitabine are currently used for the treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) in patients not only eligible for intensive chemotherapy but are also being explored in other hematologic and solid cancers. Based on their capacity to interfere with the DNA methylation machinery, these drugs are also referred to as hypomethylating agents (HMAs). As DNA methylation contributes to epigenetic regulation, azanucleosides are further considered to be among the first true "epigenetic drugs" that have reached clinical application. However, intriguing new evidence suggests that DNA hypomethylation is not the only mechanism of action for these drugs. This review summarizes the experience from more than 10 years of clinical practice with azanucleosides and discusses their molecular actions, including several not related to DNA methylation. A particular focus is placed on possible causes of primary and acquired resistances to azanucleoside treatment. We highlight current limitations for the success and durability of azanucleoside-based therapy and illustrate that a better understanding of the molecular determinants of drug response holds great potential to overcome resistance

A novel long non-coding RNA from NBL2 pericentromeric macrosatellite forms a perinucleolar aggregate structure in colon cancer

Primate-specific NBL2 macrosatellite is hypomethylated in several types of tumors, yet the consequences of this DNA hypomethylation remain unknown. We show that NBL2 conserved repeats are close to the centromeres of most acrocentric chromosomes. NBL2 associates with the perinucleolar region and undergoes severe demethylation in a subset of colorectal cancer (CRC). Upon DNA hypomethylation and histone acetylation, NBL2 repeats are transcribed in tumor cell lines and primary CRCs. NBL2 monomers exhibit promoter activity, and are contained within novel, non-polyA antisense lncRNAs, which we designated TNBL (Tumor-associated NBL2 transcript). TNBL is stable throughout the mitotic cycle, and in interphase nuclei preferentially forms a perinucleolar aggregate in the proximity of a subset of NBL2 loci. TNBL aggregates interact with the SAM68 perinucleolar body in a mirror-image cancer specific perinucleolar structure. TNBL binds with high affinity to several proteins involved in nuclear functions and RNA metabolism, such as CELF1 and NPM1. Our data unveil novel DNA and RNA structural features of a non-coding macrosatellite frequently altered in cancer

The Histone Variant MacroH2A1 Regulates Key Genes for Myogenic Cell Fusion in a Splice-Isoform Dependent Manner

Altres ajuts: This research project was supported by the Fundació La Marató de TV3 257/C/2019 (to MB). Research at the IJC is supported by the 'La Caixa' Foundation, the Fundació Internacional Josep Carreras, Celgene Spain.MacroH2A histone variants have functions in differentiation, somatic cell reprogramming and cancer. However, at present, it is not clear how macroH2As affect gene regulation to exert these functions. We have parted from the initial observation that loss of total macroH2A1 led to a change in the morphology of murine myotubes differentiated ex vivo. The fusion of myoblasts to myotubes is a key process in embryonic myogenesis and highly relevant for muscle regeneration after acute or chronic injury. We have focused on this physiological process, to investigate the functions of the two splice isoforms of macroH2A1. Individual perturbation of the two isoforms in myotubes forming in vitro from myogenic C2C12 cells showed an opposing phenotype, with macroH2A1.1 enhancing, and macroH2A1.2 reducing, fusion. Differential regulation of a subset of fusion-related genes encoding components of the extracellular matrix and cell surface receptors for adhesion correlated with these phenotypes. We describe, for the first time, splice isoform-specific phenotypes for the histone variant macroH2A1 in a physiologic process and provide evidence for a novel underlying molecular mechanism of gene regulation

Histone variant MacroH2A1 is downregulated in prostate cancer and influences malignant cell phenotype

Background: Prostate cancer (PCa), a major cause of cancer-related morbidity and mortality worldwide and mostly asymptomatic at earliest stages, is characterized by disruption of genetic and epigenetic balance. A better understanding of how those mechanisms orchestrate disease might improve diagnostic and prognostic tools, allowing for improvements in treatment efficacy. Replacement of canonical histones, an epigenetic mechanism, is highly conserved among species and altered expression of histones variants (e.g., MacroH2A1) has been associated with tumorigenesis. H2AFY gene encodes two isoforms of H2A histone variant MacroH2A1: MacroH2A1.1 and MacroH2A1.2. Specifically, MacroH2A1.1 isoform inhibits cell proliferation and promotes cellular differentiation. Because the contribution of this histone variant to carcinogenesis has been reported in several cancer types, but not for PCa, we aimed to investigate the contribution of MacroH2A1 for prostate carcinogenesis. Methods: MacroH2A1, MacroH2A1.1 and MacroH2A1.2 isoforms and the corresponding splicing regulators transcript levels were evaluated by RT-qPCR, in a tissue cohort composed by PCa, prostatic intraepithelial neoplasia (PIN) and normal prostate cases. Knockdown for MacroH2A1 and MacroH2A1.1 was performed through lentiviral transduction in DU145 cells, and MacroH2A1.1 overexpression was achieved in LNCaP cells by plasmid transfection, followed by functional assays. Biological and/or experimental replicates were performed when necessary, and specific statistical tests were applied to perform data analysis. Results: MacroH2A1.1 transcript levels were downregulated in PIN and primary PCa compared to normal prostate tissues. The same was found for QKI, a MacroH2A1.1's splicing regulator. Moreover, lower MacroH2A1.1 and QKI expression levels associated with less differentiated tumors (Gleason score ≥ 7). Interestingly, MacroH2A1.1, but more impressively DDX17 (AUC = 0.93; p < 0.0001) and QKI (AUC = 0.94; p < 0.0001), accurately discriminated cancerous from noncancerous prostate tissues. Furthermore, in PCa cell lines, total MacroH2A1 knockdown augmented malignant features, whereas MacroH2A1.1 overexpression impressively attenuated the malignant phenotype. Conclusions: Overall, our data, derived from primary PCa tissues and cell lines, anticipate a tumor suppressive role for MacroH2A1, particularly for the MacroH2A1.1 isoform, in prostate carcinogenesis

Divergent leukaemia subclones as cellular models for testing vulnerabilities associated with gains in chromosomes 7, 8 or 18

Haematopoietic malignancies are frequently characterized by karyotypic abnormalities. The development of targeted drugs has been pioneered with compounds against gene products of fusion genes caused by chromosomal translocations. While polysomies are equally frequent as translocations, for many of them we are lacking therapeutic approaches aimed at synthetic lethality. Here, we report two new cell lines, named MBU-7 and MBU-8, that differ in complete trisomy of chromosome18, a partial trisomy of chromosome 7 and a tetrasomy of the p-arm of chromosome 8, but otherwise share the same mutational pattern and complex karyotype. Both cell lines are divergent clones of U-937 cells and have the morphology and immunoprofile of monocytic cells. The distinct karyotypic differences between MBU-7 and MBU-8 are associated with a difference in the specific response to nucleoside analogues. Taken together, we propose the MBU-7 and MBU-8 cell lines described here as suitable in vitro models for screening and testing vulnerabilities that are associated with the disease-relevant polysomies of chromosome 7, 8 and 18