Refine
Year of publication
Document Type
- Doctoral Thesis (117)
Has Fulltext
- yes (117)
Is part of the Bibliography
- no (117)
Keywords
- Gentherapie (4)
- Akute lymphatische Leukämie (3)
- AF4 (2)
- Genfallen-Vektoren (2)
- Hämatopoese (2)
- LINE-1 (2)
- Primäre Immundefekte (2)
- Promotor <Genetik> (2)
- Transkriptionsfaktor (2)
- Virologie (2)
Institute
- Pharmazie (48)
- Biochemie, Chemie und Pharmazie (39)
- Biochemie und Chemie (27)
- Medizin (5)
- Biowissenschaften (1)
- Georg-Speyer-Haus (1)
- keine Angabe Institut (1)
Chromosomal translocations (CTs) are a genetic hallmark of cancer. They could be identified as recurrent genetic aberrations in hemato-malignancies and solid tumors. More than 40% of all "cancer genes" were identified in recurrent CTs. Most of these CTs result in the production of oncofusion proteins of which many have been studied over the past decades. They influence signaling pathways and/or alter gene expression. However, a precise mechanism for how these CTs arise and occur in a nearly identical fashion in individuals remains to be elucidated. Here, we performed experiments that explain the onset of CTs: proximity of genes able to produce prematurely terminated transcripts, which leads to the production of transspliced fusion RNAs, and finally, the induction of DNA double-strand breaks which are subsequently repaired via EJ repair pathways. Under these conditions, balanced chromosomal translocations could be specifically induced.
As one of the most widespread infectious diseases in the world, it is currently estimated that approximately 296 million people globally are chronically infected with Hepatitis B virus (HBV), the consequences of HBV infection cause more than 620,000 deaths each year. Although safe and effective HBV vaccines have reduced the incidence of new HBV infections in most countries, there are still around 1.5 million new infections each year. HBV remains a major health problem because there is no large-scale effective vaccination strategy in many countries with a high burden of disease, many people with chronic HBV infection are not receiving effective and timely treatment, and a complete cure for chronic infection is still far from being achieved.
Since its discovery, HBV has been identified as an enveloped DNA virus with a diameter of 42 nm. For efficient egress from host cells, HBV is thought to acquire the viral envelope by budding into multivesicular bodies (MVBs) and escape from infected cells via the exosome release pathway. It is clear that HBV hijacks the host vesicle system to complete self-assembly and propagation by interacting with factors that mediate exosome formation. Consequently, the overlap with exosome biogenesis, using MVBs as the release platform, raises the possibility for the release of exosomal HBV particles. Currently, virus containing exosomal vesicles have been described for several viruses. In light of this, this study explored whether intact HBV-virions wrapped in exosomes are released by HBV-producing cells.
First, this study established a robust method for efficient separation of exosomes from HBV virions by a combination of differential ultracentrifugation and iodixanol density gradient centrifugation. Fractionation of the density gradient revealed that two populations of infectious viral particles can be separated from the culture fluids of HBV-producing cells. The population present in the low-density peak co-migrates with the exosome markers. Whereas the population that appeared in the high-density fractions was the classical HBV virions, which are rcDNA-containing nucleocapsids encapsulated by the HBV envelope.
Subsequently, the characterization of this low-density population was performed, namely the highly purified exosome fraction was systematically investigated. Relying on the detergent sensitivity of the exosome membrane and the outer envelope of the HBV virus, disruption of the exosome structure by treatment with limited detergent revealed the presence of HBsAg in the exosomes. At the same time, mild and limited NP-40 treatment of highly purified exosomes and a further combination of density gradient centrifugation resulted in the stepwise release of intact HBV virions and naked capsids from the exosomes generated by HBV-producing cells. This implies the presence of intact HBV particles encapsulated by the host membrane.
The presence of exosome-encapsulated HBV particles was consequently also verified by suppressing the morphogenesis of MVBs or exosomes. Impairment of MVB- or exosome-generation with small molecule inhibitors has significantly inhibited the release of host membrane-encapsulated HBV particles as well. Likewise, silencing of exosome-related proteins caused a diminution of exosome output, which compromised the budding efficiency of wrapped HBV.
Moreover, electron microscopy images of ultra-thin sections combined with immunogold staining visualized the hidden virus in the exosomal structure. Additionally, the presence of LHBs on the surface of exosomes derived from HBV-expressing cells was also observed.
As expected, these exosomal membrane-wrapped HBV particles can spread productive infection in differentiated HepaRG cells. In HBV-susceptible cells, as LHBs on the membrane surface, this type of exosomal HBV appeared to be uptaken in an NTCP receptor-dependent manner.
Taken together these data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV. Exosomes hijacked by HBV act as a transporter impacting the dissemination of the virus.
Identifizierung potenzieller Taspase1 Inhibitoren für die Behandlung von t(4,11) akuter Leukämie
(2022)
Leukämie ist die häufigste bösartige Krebserkrankung im Kindes- und Jugendalter. Bei einem Kind von 1120 Kindern wird Leukämie diagnostiziert, dabei trifft diese Diagnose Jungen 30 % häufiger als Mädchen. Die Krankheitssymptome treten bei den Kindern noch vor dem Schulalter auf und am häufigsten haben die Kinder mit der akuten Form zu kämpfen. Bei einer Diagnose mit einer akuten lymphatischen Leukämie (ALL) haben die Kinder meist eine gute Prognose, während bei der akuten myeloischen Leukämie (AML) eine deutlich schlechtere.1
Die t(4;11)(q21;q23) Translokation ist aufgrund ihres häufigen Auftretens und der damit schlechten verbundenen Prognose eines der bekanntesten strukturellen Chromosomen-anomalien bei akuten Leukämien. Diese Translokation wurde das erste Mal 1977 von Oshimura et al. beschrieben.2 Bei einer t(4;11)-Translokation ist das Chromosom 4 und das Chromosom 11 involviert. Auf Chromosom 4 ist das AF4-Gen lokalisiert (AFF1) und auf dem Chromosom 11 liegt das MLL-Gen (ALL-1, HRX, hTRX, KMT2A).
Taspase1 wurde als ein proteolytisch prozessierendes Enzym identifiziert, das sich in Wirbellosen und Vertebraten zusammen mit Mitgliedern der Trithorax/MLL/KMT2A-Protein¬familie koevolviert hat. Taspase1 prozessiert nicht nur das MLL und MLL2, deren Fusions¬proteine AF4-MLL, sondern auch den Transkriptionsfaktor IIA (TFIIA) sowohl in vitro als auch in vivo.3
Die Dimerisierung von Taspase1 löst eine intrinsische Serinproteasefunktion aus, die zum katalytischen Rest Thr234 führt, der die Konsensussequenz Q-3X-2D-1•G1X2D3D4 katalysiert, die in Mitgliedern der MLL-Familie sowie im Transkriptionsfaktor TFIIA vorhanden ist. Taspase1 ist kein klassisches Enzym, da es seine Zielproteine stöchiometrisch hydrolysiert. Diese Eigenschaft macht es nahezu unmöglich, in einem klassischen Screening-Setup nach potenziellen Inhibitoren zu screenen.
In dieser Arbeit wurde ein Homogeneous time-resolved fluorescence HTRF-Reporter-Assays etabliert. Das etablierte Testsystem ermöglicht erstmalig die Untersuchung von Substanzen zusammen mit Taspase1 Monomere, die in einem zellfreien System (cfs) hergestellt wurden. Durch die Expression non monomeren Taspase1 Proteinen sollten Inhibitoren durch das etablierte Screening-Verfahren gefunden werden, die sowohl (1) Dimerisierung, (2) Autoaktivierung oder (3) Substratbindung selektiv blockieren können. Die durchgeführten Experimente führten zur Identifikation eines ersten Taspase1-Inhibitors, Closantel sodium. Closantel sodium ist ein U.S. Food and Drug Administration (FDA) zugelassenes Medikament, das Taspase1 auf nicht-kovalente Weise bindet. Die erzielten Daten zeigen, dass Closantel sodium den Dimerisierungsschritt und/oder die intrinsische Serinproteasefunktion blockiert. Closantel sodium hemmte die Spaltung des eingesetzten CS2-Substratproteins mit einem IC50 zwischen 1,6 und 3,9 µM, je nachdem, welches Taspase1-Präparat in dem HTRF Screening Assay ver¬wendeten (cfs- oder E.coli-produziert). Die Daten weisen darauf hin, dass Closantel sodium als allosterischer Inhibitor gegen die Taspase1 fungiert. Taspase1 wird zur Aktivierung der AF4-MLL-Onkofusionsproteine benötigt und wird auch in mehreren soliden Tumoren überexprimiert. Daher könnte dieser neue Inhibitor für die weitere Validierung von Taspase1 als Ziel für die Krebstherapie und für das Design potenterer Liganden für zukünftige klinische Anwendungen nützlich sein.
Standard cancer therapy research targets tumor cells while not considering the damage on the tumor microenvironment (TME) and its associated implications in impairing therapy response. Employing patients-derived organoids (PDOs) and matched stroma cells or a novel murine preclinical rectal cancer model of local radiotherapy, it was demonstrated that tumor cells-derived IL-1α polarizes cancer-associated fibroblasts towards an inflammatory (iCAFs) phenotype. While numerous studies in different tumor entities highlighted the molecular heterogeneity of CAFs, so far there are no clear findings on their functional heterogeneity and relevance in therapy resistance and response. The present study molecularly characterized iCAFs subpopulation among RCA patients as well as the preclinical mouse model and importantly unraveled the detailed molecular mechanism underlying their contribution to impair therapy response. Mechanistically, iCAFs were demonstrated to be characterized by an upregulation of nitric oxide synthase (iNOS) which triggered accumulation of reactive nitrogen species (RNS) and subsequently an oxidative DNA damage response (DDR). Such a baseline IL-1α-driven DNA damage further sensitized iCAFs to a p53-mediated therapy induced senescence (TIS) causing extensive extracellular matrix (ECM) changes and induction of senescence associated secretory phenotype (SASP) that favored tumor progression and hindered tumor cell death. Moreover, iCAFs reversibility and repolarization into more quiescent like phenotype was demonstrated upon IL-1 signaling inhibition by anakinra, a recombinant IL-1 receptor antagonist (IL1RA). Accordingly, treating mice with anakinra or specific deletion of Il1r1 in CAFs sensitized stroma-rich resistant tumors to chemoradiotherapy (CRT). Similarly, targeting CAFs senescence by senotherapy (venetoclax chemical) or employing Trp53 deficient mice reverted therapy resistance among non-responsive tumors in vivo by reducing ECM deposition and consequently favoring CD8+ T cells intratumoral infiltration posttherapy. Importantly, rectal cancer patients that do not completely respond to neoadjuvant therapy displayed an iCAFs senescence program post-CRT. Moreover, these patients presented a baseline increased CAFs content, a dominant iCAFs signature that correlated with poorer disease-free survival (DFS) and a significantly reduced circulating IL1RA serum levels. While reduced pretherapeutic IL1RN gene expression predicted poor prognosis among RCA patients, IL1RA serum levels were associated with rs4251961 (T/C) single nucleotide polymorphism (SNP) in the IL1RN gene. Finally, functional validation assays revealed that conditioned media of PDOs drove inflammatory polarization of fibroblasts and consequently rendered them sensitive to RNS-mediated DNA damage and TIS. Collectively, the study highlighted a crucial and novel role of a CAFs subset, iCAFs, in therapy resistance among RCA patients, shedding light on their functional relevance by identifying IL-1 signaling as an appealing target for their repolarization and successful targeting. Therefore, it makes sense to combine the newly demonstrated and thoroughly proven therapeutic approach of targeting IL-1 signaling in combination with conventional CRT and possibly immunotherapy. This might have a major impact on RCA therapy and be of immense relevance for other stroma-rich tumors.
The majority of B-cell precursor acute leukemias in infants are associated with the chromosomal translocation t(4;11)(q21;q23), resulting in the fusion of the mixed-lineage leukemia (MLL) and ALL1-fused gene of chromosome 4 (AF4) genes. While the fusion protein MLL-AF4 is expressed in all t(4;11) patients and essential for leukemia progression, the distinct role of the reciprocal fusion protein AF4-MLL, that is expressed in only 50-80% of t(4;11) leukemia patients (Meyer et al., 2018), remains unclear. In addition, t(4;11) leukemia could so far exclusively be generated in vivo in the presence of AF4-MLL and independent of the co-expression of MLL-AF4 (Bursen et al., 2010).
In a multifactorial approach inhibiting histone deacetylases (HDACs) and expressing the dominant negative mutation of Taspase1 (dnTASP1), both MLL fusion proteins were targeted simultaneously to evaluate a possible cooperative effect between MLL-AF4 and AF4-MLL during the progression of leukemia. Of note, neither HDACi nor dnTASP1 expression negatively affect endogenous MLL, but rather endorse its function hampered by the MLL fusion proteins (Ahmad et al., 2014; Bursen et al., 2004; Zhao et al., 2019). The mere expression of dnTASP1 failed to induce apoptosis, whereas dnTASP1 could elevate apoptosis levels significantly in HDACi-treated t(4;11) cells underlining the therapeutic potential of co-inhibiting both MLL fusion proteins.
Next, the impact of inhibiting either MLL-AF4 or AF4-MLL in vivo was resolved using whole transcriptome analysis. In PDX cells obtained by the Jeremias Laboratory (Völse, 2020) that co-expressed both t(4;11) fusion proteins, the knock-down of MLL-AF4 revealed the down-regulation of pivotal hemato-malignant factors. The expression of dnTASP1 led to massive deregulation of cell-cycle genes in vivo. Considering that the inhibition of particularly MLL-AF4 but not AF4-MLL impaired leukemic cell growth in vivo (Völse, 2020), the results of this work suggest a cooperative effect between both fusion proteins, while the loss of AF4-MLL during leukemia progression appears not essential.
Thereafter, a possible short-term role of AF4-MLL during the establishment of t(4;11) leukemia was analyzed. For this purpose, an in vitro t(4;11) model was constructed to investigate the transforming potential of transiently expressed AF4-MLL in cells constitutively expressing MLL-AF4, putatively reflecting the situation in vivo. Due to the lack of a leukemic background of the applied cell line, the aim was to investigate the long-term potential of AF4-MLL to significantly alter the epigenome rather than mimicking the development of leukemia. Strikingly, short-term-expressed AF4-MLL in cooperation with MLL-AF4 exerted durable epigenetic effects on gene transcription and chromatin accessibility. The here obtained in vitro data suggest a clonal evolutionary process initiated by AF4-MLL in a cooperative manner with MLL-AF4. Importantly, no long-term changes in chromatin accessibility could be observed by the transient expression of either MLL-AF4 or AF4-MLL alone.
All in all, considering endogenous MLL, MLL-AF4 and AF4-MLL in a targeted treatment is a promising approach for a more tailored therapy against t(4;11) leukemia, and AF4-MLL is suggested to act in a cooperative manner with MLL-AF4 especially during the development of a t(4;11) leukemia.
Leukemia is a cancer of the blood and bone marrow characterized by an uncontrolled proliferation and accumulation of abnormal white blood cells. Leukemia can be classified based on the course of the disease (acute or chronic) and the blood cell type involved (myeloid or lymphocytic), leading to four main subtypes: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Leukemia represents 2.5% of all new cancer cases per year, and survival rates in some leukemias remain low at 40%.
The bone marrow microenvironment (BMM) is a system within the bone marrow comprising cellular and acellular components, all of which play a major role in hematopoiesis, providing the physical space where hematopoietic stem cells (HSCs) reside. The BMM interacts with HSCs, offering a “niche” for those cells and in case of leukemia, the BMM has a supportive role in disease maintenance and progression by supporting Leukemia stem cells (LSCs). One of the components of the BMM are calcium ions. Calcium is the most abundant mineral in the body, a key component of bones and is released by parathyroid hormone (PTH) induced bone remodeling. Calcium ions play a role in the localization, engraftment and adhesion of normal HSC to extracellular matrix (ECM) proteins in the BMM via the calcium sensing receptor (CaSR), thereby maintaining normal hematopoiesis. In addition of a major regulator of calcium homeostasis, CaSR contribute to the development of different cancers, functioning as either tumor suppressor or oncogene, depending on the involved tissue. However, the role of CaSR and its associated pathways in the local BMM for the development of leukemia is poorly understood. We hypothesized that calcium ions released from bone, subject to a fine balance between osteoblasts and osteoclasts, and/or CaSR, contribute to development, progression and response to therapy.
We have shown that the local calcium concentration forms a gradient in the bone marrow niche and in mice with CML is similarly low as in control mice, but significantly higher in mice suffering from BCR ABL1 driven B ALL or MLL AF9 driven AML. Similarly, the calcium concentration in the human BMM was found to be higher in AML than in other leukemias. Regarding the function of calcium in leukemia cells, we found that AML and CML cells respond differently to calcium exposure, with AML cells exhibiting regulation of cellular processes such as adhesion to the ECM protein fibronectin and migration toward CXCL 12, whereas CML cells remained mostly unaltered. Using genetic deletion or overexpression of CaSR in murine models of leukemia, we observed that CaSR acts as tumor suppressor in BCR-ABL1 driven CML and B ALL and as oncogene in AML.
Focusing on AML, our data shows that deficiency of CaSR on LICs leads, on one hand to increased apoptosis, and on the other hand to reduced cell cycle, reactive oxygen species (ROS) production and DNA damage in vivo, which may explain the observed prolongation of survival of mice. Complementary, in vitro experiments demonstrated that cells overexpressing CaSR have a distinct, cancer promoting phenotype compared to wildtype cells. Overexpression of CaSR led to an increase in proliferation, cell cycle, ROS production, DNA damage and reduced apoptosis. We have identified CaSR mediated pathways in AML and shown that CaSR enhances leukemia progression by activating MAPK/ERK and Wnt β catenin signaling. In addition, the CaSR interacting protein filamin A (FLNA) was shown to contribute to aggressive disease in vitro and in vivo. Furthermore, the mechanism underlying the role of CaSR in AML pathogenesis and possible regulation of LSCs was studied. Our findings demonstrated that CaSR ablation reduces myeloid progenitor function and proved that CaSR is required for maintenance of LSC pool by regulating its frequency and function. Further supporting the role of CaSR in LSC maintenance, genes associated with AML stemness and self renewal capacity were upregulated when CaSR was overexpressed and downregulated when CaSR was depleted. Given the role of CaSR in AML, the CaSR antagonist NPS 2143 was tested in vivo. The combination treatment of NPS 2143 with the standard of care, ara C, significantly reduced the tumor burden and prolonged the survival of mice with AML in syngeneic and xenotransplantation experiments. Based on the finding that CaSR functions as a tumor suppressor in CML, treatment of mice with the CaSR agonist cinacalcet in combination with imatinib prolonged survival of mice with CML compared to treatment with the mice given vehicle.
Our results suggest that calcium ions stemming from the calcium-rich BMM via CaSR strongly and differentially influence leukemia progression. As an adjunct to existing treatment therapies, targeting of CaSR with specific pharmacologic antagonists may prolong survival of patients with AML.
Chapter I of this work addressed the piggyBac (PB) transposon system, a non-viral genome engineering tool that is capable of efficiently performing stable integration of DNA sequences into a target cells genome and has already been used in clinical trials. However, the PB transposase has the problematic property of preferentially integrating transposons near transcriptional start sites (TSSs). This increases the likelihood of causing genotoxic effects, limiting its potential use as a tool in clinical applications. It has been shown in the past that the PB transposase shows physical interactions with BET proteins (e.g. BRD4) through Co-IP experiments. Representatives of these proteins are part of the transcriptional activation complex and are abundant at TSSs. Accordingly, it was previously proposed that this interaction is the underlying cause for the biased integration preference. For the first chapter of this thesis, the goal was to disrupt this interaction potentially modifying said integration preference. A secondary structure hypothesized to be mainly responsible for said interaction was extensively mutated resulting in several PB variants that were analyzed for their interaction capacity through a series of Co-IP experiments with BRD4. In total, seven substitutions were identified (E380F, V390K, T392Y, M394R, K407C, K407Q, and K407V) which exhibited reduced interaction capacity with BRD4. Each of the aforementioned mutants were used to generate integration libraries and, through NGS, it was determined if the integration preferences of the respective mutants had changed. In the immediate range 200 base pairs up- and downstream from known TSSs all mutants used exhibited a reduced integration bias. At a wider observation window 3 kbp up- and downstream from TSSs, further mutants with the substitutions M394R, T392Y and V390K showed a reduction in integration frequency of 17.3%, 1.5% and 5.4%, respectively, compared to the wildtype. Of particular note was the M394R mutant, which showed a reduction in all window sizes analyzed with a maximum of 65% less integration preference in the immediate vicinity of TSSs, theoretically generating a safety advantage over the wildtype transposase.
Chapter II was dedicated to the overall safety improvement for transposon-based gene modification and addresses the time point after the transgene has already been integrated and serious side effects may not be preventable. With this in mind, the aim was to develop a novel suicide-switch that can be stably introduced into cells via transposition, and reliably leads to cell death of the modified cells once activated. A system based on CRISPR/Cas9 was developed, where single guide RNAs were used to guide the Cas9 nuclease to Alu elements. These are short, repetitive sequences, which are distributed over the human genome in more than one million copies. Inducing double strand breaks within these elements would lead to genomic fragmentation and cell death. To be inducible, a transcriptional as well as post- translational control mechanism was added. Transcription of the Cas9 nuclease was regulated using a tet-on system, making expression dependent on doxycycline (DOX) supplementation. Furthermore, a version of the Cas9 nuclease called arC9 was used that allows double strand break generation only in the presence of 4-Hydroxytamoxifen (4-HT). Together with an expression cassette for the Alu-specific guide RNA and an expression cassette for the reverse tetracycline controlled transactivator all components were arranged between transposase-specific recognition sequences on a plasmid to allow transposon-system based gene transfer. The system was tested in HeLa cells. First, conditional expression of the arC9 nuclease was confirmed by addition of 1 μg/ml DOX. Second, the suicide-switch was further induced by adding 200 nM 4-HT and protein extracts were assayed for the KAP1 phosphorylation. Only upon induction with DOX and 4-HT phosphorylated KAP1 was detected, indicating DNA damage. Further, extensive growth and survival experiments were conducted to determine the effect of suicide-switch induction on cell proliferation and survival. Between 24 and 48 hours after induction, a halt in cell division was detected, after which extensive cell death was observed. Within 5 days post induction, >99% of all cells were eliminated. In the absence of both inducers, no significant differences in survival were observed compared to control cells line lacking Alu-specific guide RNAs. Microscopic examinations of the <1% surviving cell fraction revealed a senescence-associated phenotype and showed no signs of resumption of the cell division process. Accordingly, the second chapter of this thesis also achieved its goal in developing a functional suicide-switch that can be inserted into human cells via transposition, is highly dependent on the necessary induction signals, and exhibits excellent elimination capabilities in the context tested.
KMT2A-rearrangements are causative for 70-80% all infant acute lymphoblastic leukemias (Pieters et al., 2019, 2007). Among these, the translocation t(4;11)(q21;23) generating the oncogenic fusion genes KMT2A::AFF1 and AFF1::KMT2A is the most frequent one, accounting for almost every second case of KMT2A-r infant ALL (Meyer et al., 2018). Despite passing a multimodal chemotherapy, 64% of patients achieve an event including relapse or death within four years from diagnosis, and overall survival three years from relapse remains poor with only 17% (Driessen et al., 2016; Pieters et al., 2019, 2007). Vari-ous studies have shown that relapse and therapy resistance were not mediated by chemotherapy-induced mutagenesis as there was no accumulation of secondary mutations in the dominant leukemic clone between diagnosis and relapse (Agraz-Doblas et al., 2019; Andersson et al., 2015; Bardini et al., 2011; Dobbins et al., 2013; Driessen et al., 2013; Mullighan et al., 2007).
Intriguingly, exclusively infant t(4;11) ALL patients were reported to subdivide in two groups depending on the level of HOXA gene cluster expression (Trentin et al., 2009). The HOXAlo group displayed a high expression of IRX1 and the HOXAhi group a low expression of IRX1 (Symeonidou and Ottersbach, 2021; Trentin et al., 2009). Importantly, the HOXAlo/IRX1hi group was characterized to possess a strongly ele-vated relapse incidence compared to the HOXAhi/IRX1lo group (Kang et al., 2012; Stam et al., 2010). IRX1 was identified to upregulate the Early growth response genes EGR1, EGR2 and EGR3 (Kühn et al., 2016).
The doctoral project “EGR-mediated relapse mechanisms in infant t(4;11) acute lymphoblastic leuke-mia” aimed to investigate a potential correlation between the HOXAlo-IRX1-EGR axis and relapse development in infant t(4;11) ALL. The primary objective was to clarify through which molecular mechanism(s) relapse development despite continuous chemotherapy could be achieved. In this context, the role of the EGR genes has been investigated. In addition, this project aimed to disclose molecular targets which could offer novel therapeutic interventions to interfere with therapy resistance and relapse formation.
The vascular endothelium is a monolayer of endothelial cells that builds the inner lining of the blood vessels and constitutes a regulatory organ within the physiological system to sustain homeostasis. Endothelial cells participate in physiological processes including inflammation and angiogenesis. Dysregulation of these processes, however, can evoke or maintain pathological disorders, including cardiovascular and chronic inflammatory diseases or cancer. Although pathological inflammation and angiogenesis represent treatable conditions, current pharmacotherapeutic approaches are frequently not satisfying since their long-term application can evoke therapy resistance and thus reduced clinical efficacy. Consequently, there is an ongoing demand for the discovery of new therapeutic targets and drug leads. Considering that endothelial cells play a critical role in both angiogenesis and inflammation, the vascular endothelium represents a promising target for the treatment of diseases.
Vioprolide A is a secondary metabolite isolated from the myxobacterium Cystobacter violaceus Cb. vi35. Recently, vioprolide A was identified to interact with NOP14, a nucleolar protein involved in ribosome biogenesis. Ribosome biogenesis is an indispensable cellular event that ensures adequate homeostasis. Abnormal alterations in the ribosome biogenesis, referred to as ribosomopathies, however, can lead to an overall increase in the risk of developing cancer. Accordingly, several studies have outlined the involvement of NOP14 in cancer progression and metastasis, and vioprolide A has been demonstrated to exert anti-cancer effects in vitro. However, the impact of vioprolide A and NOP14 on the endothelium has been neglected so far, although endothelial cells are crucially involved in inflammation and angiogenesis under both physiological and pathological conditions.
In the present study, the effect of vioprolide A on inflammatory and angiogenic actions was analysed. In vivo, the laser-induced choroidal neovascularization (CNV) assay outlined a strong inhibitory effect of vioprolide A on both inflammation and angiogenesis. Furthermore, intravital microscopy of the cremaster muscle in mice revealed that vioprolide A strongly impaired the TNF-induced leukocyte-endothelial cell interaction in vivo.
In further experiments, the specific effect of vioprolide A on activation processes of primary human umbilical vein endothelial cells (HUVECs) was examined. According to the in vivo results, vioprolide A decreased the leukocyte-endothelial cell interaction in vitro through downregulating the cell surface expression and total protein expression of ICAM-1, VCAM-1 and E-selectin. Vioprolide A evoked its anti-inflammatory actions via a dual mechanism: On the one hand, the expression of pro-inflammatory proteins, including TNFR1 and cell adhesion molecules, was lowered through a general downregulation of de novo protein synthesis. The inhibition of de novo protein synthesis is most likely linked to the interaction with and inhibition of NOP14 by vioprolide A in HUVECs. On the other hand, the natural product prevented the nuclear translocation and promotor activity of the pro-inflammatory transcription factor NF-ĸB. Interestingly, most anti-inflammatory compounds that interfere with the NF-ĸB signaling pathway prevent NF-ĸB nuclear translocation through recovering or stabilizing the inhibitory IĸB proteins. Vioprolide A, however, decreased rather than stabilized the IĸB proteins and prevented NF-ĸB nuclear translocation through interfering with its importin-dependent nuclear import. By performing siRNA-mediated knockdown experiments, we evaluated the role of NOP14 in inflammatory processes in HUVECs and could establish a causal link between the anti-inflammatory actions of vioprolide A and the deletion of NOP14.
Besides exerting anti-inflammatory actions, we found that vioprolide A potently decreased the angiogenic key features proliferation, migration and sprouting of endothelial cells. Mechanistically, the natural product interfered with pro-angiogenic signaling pathways. Vioprolide A reduced the protein level of growth factor receptors, including VEGFR2, which is the most prominent receptor responsible for angiogenic signaling in endothelial cells. This effect was based on the general inhibition of de novo protein synthesis by the natural product. Downregulation of growth factor receptors impaired the activation of downstream signaling intermediates, including the MAPKs ERK, JNK and p38. To our surprise, however, activation of Akt, another downstream effector of VEGFR2, was increased rather than decreased. Furthermore, vioprolide A lowered the nuclear translocation of the transcriptional coactivator TAZ, which is regulated by the evolutionary conserved Hippo signaling pathway. Interestingly, however, and in contrast to NF-ĸB, TAZ nuclear translocation in mammalian cells seems to be independent of importins. In this context, we found that vioprolide A reduced both the protein level and nuclear localization of MAML1, which is needed to retain TAZ in the nucleus after its successful translocation.
...