The central role of the transcriptional regulator IκBζ in psoriasis Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Anne Müller aus Finsterwalde Tübingen 2020 Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen. Tag der mündlichen Qualifikation: 10.11.2020 Stellvertretender Dekan: Prof. Dr. József Fortágh 1. Berichterstatter: Prof. Dr. Klaus Schulze-Osthoff 2. Berichterstatter: Prof. Dr. Alexander N.R. Weber 2 Table of Content Abbreviations .............................................................................................................. 4 Zusammenfassung ..................................................................................................... 6 Abstract ...................................................................................................................... 7 1 Introduction .............................................................................................................. 8 1.1 The pathogenesis of psoriasis ........................................................................... 8 1.2 The NF-κB pathway ......................................................................................... 13 1.3 The atypical IκB member IκBζ ......................................................................... 15 1.4 IκBζ in psoriasis ............................................................................................... 17 1.5 Project objectives ............................................................................................ 18 2 Results & Discussion ............................................................................................. 19 2.1 STAT3 and NF-κB mediate the induction of IκBζ in keratinocytes ................... 19 2.2 IκBζ regulates a subset of IL-36-dependent target genes in two waves .......... 21 2.3 IL-36-mediated dermatitis: IκBζ function in vivo .............................................. 23 2.4 Targeting IκBζ in keratinocytes as a new therapy approach for psoriasis ....... 25 2.5 Initiation of the CDK4/6-EZH2-STAT3 pathway ............................................... 26 2.6 Does CDK4/6 regulate PRC2-independent functions of EZH2? ...................... 28 2.7 CDK4/6- or EZH2-mediated inhibition of pSTAT3 at Y705 .............................. 30 2.8 Prevention of psoriasis and therapeutic application of novel inhibitors ............ 31 2.9 Working model: IκBζ induction in psoriasis...................................................... 34 3 References ............................................................................................................ 36 4 Appendix ................................................................................................................ 44 Acknowledgements ..................................................................................................... I Contributions .............................................................................................................. II 3 Abbreviations AMP Antimicrobial peptide ATP Adenosine triphosphate BAFFR B-cell activating factor receptor BCL-2 B-cell lymphoma 2 CAK CDK-activating kinase cAMP Cyclic adenosine monophosphate CARD Caspase recruitment domain CBX Chromobox CCL CC-chemokine ligand CCL20 CC-chemokine ligand 20 CD40 Cluster of differentiation 40 CDK Cyclin-dependent kinase CpG Cytosine-phosphatidyl-guanine CXCL C-X-C motif chemokine DNA Deoxyribonucleic acid EED embryonic ectoderm development ERα Estrogen receptor alpha EZH2 Enhancer of zeste homolog 2 GWAS Genome-wide association study H3 Histone 3 HAT Histone acetyltransferase HDAC Histone deacetylase ICAM Intercellular adhesion molecule IKK IκB kinase IL Interleukin IMQ Imiquimod INK inhibitor of CDK IRAK Interleukin-1 receptor-associated kinase IκB Inhibitor of NF-κB JAK Janus kinase KO Knockout LCN2 Lipocalin-2 LPS Lipopolysaccharide LTβR Lymphotoxin β receptor MALT Mucosa-associated lymphoid tissue MAPK Mitogen-activated protein kinase mDC Myeloid dendritic cells MHC Major histocompatibility complex mRNA Messenger ribonucleic acid MyD88 Myeloid differentiation primary response 88 NEMO NF-kappa-B essential modulator NF-κB Nuclear factor of kappa light chain gene enhancer in B cells NIK NF-κB inducing kinase 4 NLS Nuclear localisation sequence PCGF Polycomb group RING finger protein pDC Plasmacytoid dendritic cells PHC Polyhomeotic-like protein PRC Polycomb repressive complex RANK Receptor activator of NF-κB Rb Retinoblastoma RbAp Retinoblastoma protein associated protein RHD Rel homology domain RNF2 Ring finger protein 2 ROR RAR-related orphan receptor SNP Single nucleotide polymorphism STAT Signal transducers and activator of transcription SWI/SNF Switch/sucrose nonfermenting TAD Transactivating domain TCF T-cell factor Th17 T-helper cells 17 TLR Toll-like receptor TNF Tumor necrosis factor TRAF TNF receptor associated factor VCAM Vascular cell adhesion molecule VEGF Vascular endothelial growth factor 5 Zusammenfassung IκBζ gehört zur Gruppe der atypischen NF-κB Inhibitoren (IκBs). Im Gegensatz zu klassischen IκBs wird IκBζ fast ausschließlich induzierbar im Zellkern exprimiert, wo es die Expression von bestimmten Targetgenen nicht nur inhibieren, sondern auch aktivieren kann. Das Gen NFKBIZ, welches für IκBζ kodiert, wurde als Risikogen in der Psoriasis identifiziert. Ebenso sind IκBζ-Knockout-Mäuse in bestimmten Modellen gegenüber einer experimentellen Psoriasis geschützt. Ziel dieser Dissertation war es, die Rolle von IκBζ in der Psoriasis näher zu beleuchten, um daran anknüpfend neue Therapieoptionen zu entwickeln. Es konnte gezeigt werden, dass IκBζ neben dem IL-17 Signalweg auch den IL-36 Signalweg reguliert, welcher bei bestimmten Formen der Psoriasis eine wichtige Rolle spielt. Die IL-36-vermittelte Induktion von IκBζ wird von NF-κB und STAT3 gesteuert und führt zur Expression von bestimmten pro-inflammatorischen Genen, welche die Pathogenese der Psoriasis initiieren. Vice versa sind IκBζ- Knockout-Mäuse gänzlich vor einer IL-36-vermittelten experimentellen Psoriasis geschützt. Demnach stellt IκBζ einen zentralen Regulator in der Psoriasis dar, welcher unabhängig von der Art des Stimulus die Inflammation fördert. Darauf aufbauend folgten Screenings für pharmakologische Inhibitoren, welche die Induktion von IκBζ unterdrücken. Dabei konnte ein neuer proinflammatorischer Signalweg identifiziert werden, bei dem EZH2, eine Methyltransferase, durch CDK4/6 phosphoryliert wird und ihrerseits STAT3 methyliert, welches die IκBζ-Expression induziert. Die pharmakologische Hemmung von CDK4/6 oder EZH2 hemmte die Pathogenese von Psoriasis in vitro und in vivo. Zusammenfassend konnte diese Arbeit IκBζ nicht nur als verantwortlichen Mediator in der Psoriasis näher charakterisieren, sondern mit dem CDK4/6-EZH2-vermittelten Signalweg auch einen neuen Mechanismus identifizieren, dessen Inhibition eine mögliche Therapieoption in der Psoriasis darstellt. 6 Abstract IκBζ belongs to the group of atypical NF-κB inhibitors (IκBs). In contrast to classical IκBs, IκBζ is only inducibly expressed in the cell nucleus, where it can inhibit, but more importantly, also activate the expression of a particular subset of target genes. NFKBIZ, the gene encoding IκBζ, has been identified as new risk gene in psoriasis. Moreover, IκBζ knockout mice are protected in certain models of experimental psoriasis. The aim of this thesis was to examine the global role of IκBζ in psoriasis in order to find a new therapy approach. It could be shown that IκBζ is a regulator not only of IL-17 but also of IL-36 signaling, which plays a major role in certain forms of psoriasis. The IL-36-mediated induction of IκBζ was driven by NF-κB and STAT3, and led to the expression of pro-inflammatory genes that initiate the development of psoriasis. Accordingly, IκBζ knockout mice were completely protected from IL-36- mediated experimental psoriasis. Thus, IκBζ represents a central regulator in psoriasis, which promotes inflammation regardless of the type of stimulus. Based on this finding, screenings for small-molecule inhibitors were performed that are able to repress the induction of IκBζ. Thereby, I could identify a new pro-inflammatory signal pathway in keratinocytes. In this pathway, CDK4/6 phosphorylated the methyltransferase EZH2, which in turn methylated and activated STAT3, which transcriptionally induced IκBζ. The pharmacological inhibition of CDK4/6 or EZH2 inhibited the pathogenesis of psoriasis in vitro and in vivo. In conclusion, this thesis not only validates IκBζ as an essential mediator of psoriasis, but also identifies the CDK4/6-EZH2 axis as a novel mechanism whose inhibition could provide a potential therapeutic option for the treatment of psoriasis. 7 1 Introduction 1.1 The pathogenesis of psoriasis Psoriasis is a mixed autoinflammatory and autoimmune disease of the skin which partially develops due to genetic predispositions, but also due to a variety of environmental factors. It is characterized by scaly, erythematous patches, papules, and plaques that are often pruritic [1]. Around 2-3% of the worldwide population suffer from psoriasis, which affects statistically more women [2]. The most severe forms of psoriasis are psoriasis vulgaris (also named plaque psoriasis), inverse psoriasis, guttate psoriasis, erythrodermic psoriasis, and pustular psoriasis. Psoriasis is often associated with further