Epigenetic Anti-Cancer Treatment With a Stabilized Carbocyclic Decitabine Analogue

5-Aza-2'-deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2'-deoxycytidine (dC) to 5-methyl-dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM-13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti-proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off-target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis.

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis

Epstein-Barr-Virus-Induced One-Carbon Metabolism Drives B Cell Transformation.

Epstein-Barr virus (EBV) causes Burkitt, Hodgkin, and post-transplant B cell lymphomas. How EBV remodels metabolic pathways to support rapid B cell outgrowth remains largely unknown. To gain insights, primary human B cells were profiled by tandem-mass-tag-based proteomics at rest and at nine time points after infection; >8,000 host and 29 viral proteins were quantified, revealing mitochondrial remodeling and induction of one-carbon (1C) metabolism. EBV-encoded EBNA2 and its target MYC were required for upregulation of the central mitochondrial 1C enzyme MTHFD2, which played key roles in EBV-driven B cell growth and survival. MTHFD2 was critical for maintaining elevated NADPH levels in infected cells, and oxidation of mitochondrial NADPH diminished B cell proliferation. Tracing studies underscored contributions of 1C to nucleotide synthesis, NADPH production, and redox defense. EBV upregulated import and synthesis of serine to augment 1C flux. Our results highlight EBV-induced 1C as a potential therapeutic target and provide a new paradigm for viral onco-metabolism

Human TH17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation

It has been shown that innate immune responses can adopt adaptive properties such as memory. Whether T cells utilize innate immune signaling pathways to diversify their repertoire of effector functions is unknown. Gasdermin E (GSDME) is a membrane pore-forming molecule that has been shown to execute pyroptotic cell death and thus to serve as a potential cancer checkpoint. In the present study, we show that human T cells express GSDME and, surprisingly, that this expression is associated with durable viability and repurposed for the release of the alarmin interleukin (IL)-1α. This property was restricted to a subset of human helper type 17 T cells with specificity for Candida albicans and regulated by a T cell-intrinsic NLRP3 inflammasome, and its engagement of a proteolytic cascade of successive caspase-8, caspase-3 and GSDME cleavage after T cell receptor stimulation and calcium-licensed calpain maturation of the pro-IL-1α form. Our results indicate that GSDME pore formation in T cells is a mechanism of unconventional cytokine release. This finding diversifies our understanding of the functional repertoire and mechanistic equipment of T cells and has implications for antifungal immunity

Die Rolle von NFκB für die Aufnahme von Glucose und das Überleben von B-Zell-Lymphomen

Continuously proliferating cells must exhibit high levels of nutrient uptake in order to support biosynthetic pathways. Most cancer cells exhibit elevated glucose import and consumption rates when compared with normal tissue. This work investigates the signals that govern glucose uptake in B-cell lymphomas. The experiments presented provide evidence that the NFκB pathway controls glucose import via GLUT1 membrane trafficking in both viral and spontaneous B-cell lymphomas. To promote GLUT1 translocation the NFκB pathway activates the signaling cascade from PI3K to GLUT1 plasma membrane translocation at two distinct points. First, the NFκB-stimulating IκB-kinase β (IKKβ) supported Phosphatidylinositol-3-kinase (PI3K) driven AKT activity. Second, NFκB-induced transcription was required for GLUT1 surface localization downstream of AKT, by licensing AKT-mediated phosphorylation of the GLUT1 regulator, AKT Substrate of 160kD (AS160). The requirement for NFκB was specific for productive AKT/AS160 interaction, as AKT phosphorylation of the mammalian target of Rapamycin (mTOR) regulator Tuberous Sclerosis 2 (TSC2) was independent of NFκB. In Epstein Barr virus (EBV) transformed B-cells the NFκB pathway was essential to sustain energy homeostasis and survival. Inhibition of NFκB-induced transcription repressed glucose uptake and glycolytic flux leading to starvation and the induction of caspase-independent cell death associated with autophagy. Alternate carbon sources reduced cell death and autophagy induction after NFκB inhibition, underlining the impact of starvation on cell death. In contrast, inhibitors of autophagy and oxidative phosphorylation specifically accelerated cell death after NFκB inhibition. Thus, autophagy and mitochondrial metabolism serve as pro-survival pathways after NFκB inhibition. Small molecule-mediated IKKβ inhibition in diffuse large B-cell lymphomas, EBV transformed B-cells and Karposi sarcoma herpesvirus infected primary effusion lymphomas induced starvation, autophagy, and rendered cells more sensitive to inhibitors mitochondrial metabolism. Thus, IKKβi mimics the metabolic phenotype observed in NFκB-inhibited LCLs. Taken together, the results of this work suggest that NFκB signaling establishes a metabolic program supporting proliferation and apoptosis resistance by driving glucose import. The combined targeting of NFκB pathway and mitochondrial metabolism might be a valid synthetic lethal approach to treat B-cell malignancies.Zellen, die sich ununterbrochen teilen, müssen ausreichend Nährstoffe aufnehmen, um die notwendigen Biosynthesereaktionen durchführen zu können. Viele Krebszellen weisen daher einen erhöhten Glucosestoffwechsel auf. Die vorliegende Doktorarbeit untersucht Signalwege, die den Stoffwechsel in B-Zell-Lymphomen regulieren. Die ermittelten Daten legen nahe, dass der NFκB Signalweg den Import von Glucose in spontanen und virusinduzierten B-Zell-Lymphomen verstärkt, indem er die Translokation des Glucosetransporters 1 (GLUT1) von cytoplasmatischen Vesikeln zur Plasmamembran ermöglicht. Dabei aktivierte NFκB die Signalkaskade zwischen Phosphatidylinositol-3-kinase (PI3K) und AKT, die für die Translokation von GLUT1 verantwortlich ist, an zwei unterschiedlichen Stellen. Einerseits verstärkte die IκB-kinase β (IKKβ) die Wirkung von PI3K auf AKT, andererseits ist NFκB-induzierte Transkription notwendig, damit AKT den GLUT1 Regulator AKT substrate of 160kDA (AS160) phosphorylieren kann. Da die AKT-abhängige Phosphorylierung von Tuberous Sclerosis 2 (TSC2) im mammalian target of Rapamycin (mTOR) Signalweg unabhängig von der NFκB-induzierten Transkription war, ist NFκB speziell für die Phosphorylierung von AS160 nötig. In Epstein Barr Virus (EBV) transformierten B-Zellen ist der NFκB Signalweg für die Energiehomöostase und das Überleben notwendig. Das Ausschalten der NFκB-induzierten Transkription unterdrückte die Aufnahme von Glucose, verminderte den Stoffumsatz in der Glycolyse und verursachte einen caspaseunabhängigen Zelltod mit Merkmalen von Autophagie. Glutamin und α-Ketoglutarat, die als alternative Nährstoffe für den Mitochondrienstoffwechsel fungieren, verringerten Zelltod und Autophagie nach dem Verlust von NFκB-induzierter Transkription. Im Gegensatz dazu führte die pharmakologische Inhibierung von Autophagie oder oxidativer Phosphorylierung zu einem beschleunigten Zelltod nach dem Verlust von NFκB-induzierter Transkription. Folglich sind Autophagie und Mitochondrienstoffwechsel wichtig für das Überleben von NFκB-inhibierten Zellen. Die pharmakologische Inhibierung von IKKβ in diffusen großzelligen B-Zell-Lymphomen, EBV-transformierten B-Zellen und Kaposi-Sarkom-Herpesvirus infizierten Primary Effusion Lymphomas verursachte eine Internalisierung von GLUT1, verstärkte Autophagie und die Abhängigkeit vom Mitochondrienstoffwechsel. Die Inhibierung von IKKβ führte damit zum gleichen Stoffwechselphänotyp wie der Verlust von NFκB-induzierter Transkription. Zusammengefasst zeigen die Ergebnisse dieser Arbeit, dass der NFκB Signalweg ein Stoffwechselprogramm in B-Zellen etabliert, welches durch eine erhöhte Glucoseaufnahme Teilung und Überleben der Zelle unterstützt. Die gewonnenen Erkenntnisse könnten einen ersten Ansatz für die Entwicklung einer kombinierten Therapie für B-Zell-Lymphome darstellen, bei der sowohl der NFκB Signalweg als auch der Mitochondrienstoffwechsel blockiert werden

Catalytic Ferrier rearrangement of unsaturated nucleosides

The attempted intermolecular addition of malonyl radicals to 1',2'- unsaturated nucleosides has led to the unexpected formation of furanones. Thus, only catalytic amounts of ceric(IV) ammonium nitrate (CAN), induce a Ferrier rearrangement. The unsaturated lactone was isolated in good yield and can serve as a precursor for the synthesis of optically active products