@phdthesis{Gruendl2021,
  author    = {Gr{\"u}ndl, Marco},
  title     = {Biochemical characterization of the MMB-Hippo crosstalk and its physiological relevance for heart development},
  doi       = {10.25972/OPUS-21332},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213328},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The Myb-MuvB (MMB) complex plays an essential role in the time-dependent transcriptional activation of mitotic genes. Recently, our laboratory identified a novel crosstalk between the MMB-complex and YAP, the transcriptional coactivator of the Hippo pathway, to coregulate a subset of mitotic genes (Pattschull et al., 2019). Several genetic studies have shown that the Hippo-YAP pathway is essential to drive cardiomyocyte proliferation during cardiac development (von Gise et al., 2012; Heallen et al., 2011; Xin et al., 2011). However, the exact mechanisms of how YAP activates proliferation of cardiomyocytes is not known. This doctoral thesis addresses the physiological role of the MMB-Hippo crosstalk within the heart and characterizes the YAP-B-MYB interaction with the overall aim to identify a potent inhibitor of YAP. The results reported in this thesis indicate that complete loss of the MMB scaffold protein LIN9 in heart progenitor cells results in thinning of ventricular walls, reduced cardiomyocyte proliferation and early embryonic lethality. Moreover, genetic experiments using mice deficient in SAV1, a core component of the Hippo pathway, and LIN9-deficient mice revealed that the correct function of the MMB complex is critical for proliferation of cardiomyocytes due to Hippo-deficiency. Whole genome transcriptome profiling as well as genome wide binding studies identified a subset of Hippo-regulated cell cycle genes as direct targets of MMB. By proximity ligation assay (PLA), YAP and B-MYB were discovered to interact in embryonal cardiomyocytes. Biochemical approaches, such as co-immunoprecipitation assays, GST-pulldown assays, and µSPOT-based peptide arrays were employed to characterize the YAP-B-MYB interaction. Here, a PY motif within the N-terminus of B-MYB was found to directly interact with the YAP WW-domains. Consequently, the YAP WW-domains were important for the ability of YAP to drive proliferation in cardiomyocytes and to activate MMB target genes in differentiated C2C12 cells. The biochemical information obtained from the interaction studies was utilized to develop a novel competitive inhibitor of YAP called MY-COMP (Myb-YAP competition). In MY-COMP, the protein fragment of B-MYB containing the YAP binding domain is fused to a nuclear localization signal. Co-immunoprecipitation studies as well as PLA revealed that the YAP-B-MYB interaction is robustly blocked by expression of MY-COMP. Adenoviral overexpression of MY-COMP in embryonal cardiomyocytes suppressed entry into mitosis and blocked the pro-proliferative function of YAP. Strikingly, characterization of the cellular phenotype showed that ectopic expression of MY-COMP led to growth defects, nuclear abnormalities and polyploidization in HeLa cells. Taken together, the results of this thesis reveal the mechanism of the crosstalk between the Hippo signaling pathway and the MMB complex in the heart and form the basis for interference with the oncogenic activity of the Hippo coactivator YAP.},
  subject      = {Zellzyklus},
  language  = {en}
}
@phdthesis{Simon2019,
  author    = {Simon, Katja},
  title     = {Identifying the role of Myb-MuvB in gene expression and proliferation of lung cancer cells},
  doi       = {10.25972/OPUS-16181},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161814},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The evolutionary conserved Myb-MuvB (MMB) multiprotein complex is a transcriptional master regulator of mitotic gene expression. The MMB subunits B-MYB, FOXM1 as well as target genes of MMB are often overexpressed in different cancer types. Elevated expression of these genes correlates with an advanced tumor state and a poor prognosis for patients. Furthermore, it has been reported that pathways, which are involved in regulating the mitotic machinery are attractive for a potential treatment of cancers harbouring Ras mutations (Luo et al., 2009). This suggest that the MMB complex could be required for tumorigenesis by mediating overactivity of mitotic genes and that the MMB could be a useful target for lung cancer treatment. However, although MMB has been characterized biochemically, the contribution of MMB to tumorigenesis is largely unknown in particular in vivo. In this thesis, it was demonstrated that the MMB complex is required for lung tumorigenesis in vivo in a mouse model of non small cell lung cancer. Elevated levels of B-MYB, NUSAP1 or CENPF in advanced tumors as opposed to low levels of these proteins levels in grade 1 or 2 tumors support the possible contribution of MMB to lung tumorigenesis and the oncogenic potential of B-MYB.The tumor growth promoting function of B-MYB was illustrated by a lower fraction of KI-67 positive cells in vivo and a significantly high impairment in proliferation after loss of B-Myb in vitro. Defects in cytokinesis and an abnormal cell cycle profile after loss of B-Myb underscore the impact of B-MYB on proliferation of lung cancer cell lines. The incomplete recombination of B-Myb in murine lung tumors and in the tumor derived primary cell lines illustrates the selection pressure against the complete loss of B-Myb and further demonstrats that B-Myb is a tumor-essential gene. In the last part of this thesis, the contribution of MMB to the proliferation of human lung cancer cells was demonstrated by the RNAi-mediated depletion of B-Myb. Detection of elevated B-MYB levels in human adenocarcinoma and a reduced proliferation, cytokinesis defects and abnormal cell cycle profile after loss of B-MYB in human lung cancer cell lines underlines the potential of B-MYB to serve as a clinical marker.},
  subject      = {Lungenkrebs},
  language  = {en}
}
@phdthesis{WeinstockgebPattschull2019,
  author    = {Weinstock [geb. Pattschull], Grit},
  title     = {Crosstalk between the MMB complex and YAP in transcriptional regulation of cell cycle genes},
  doi       = {10.25972/OPUS-17086},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170866},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The Myb-MuvB (MMB) multiprotein complex is a master regulator of cell cycle-dependent gene expression. Target genes of MMB are expressed at elevated levels in several different cancer types and are included in the chromosomal instability (CIN) signature of lung, brain, and breast tumors. This doctoral thesis showed that the complete loss of the MMB core subunit LIN9 leads to strong proliferation defects and nuclear abnormalities in primary lung adenocarcinoma cells. Transcriptome profiling and genome-wide DNA-binding analyses of MMB in lung adenocarcinoma cells revealed that MMB drives the expression of genes linked to cell cycle progression, mitosis, and chromosome segregation by direct binding to promoters of these genes. Unexpectedly, a previously unknown overlap between MMB-dependent genes and several signatures of YAP-regulated genes was identified. YAP is a transcriptional co-activator acting downstream of the Hippo signaling pathway, which is deregulated in many tumor types. Here, MMB and YAP were found to physically interact and co-regulate a set of mitotic and cytokinetic target genes, which are important in cancer. Furthermore, the activation of mitotic genes and the induction of entry into mitosis by YAP were strongly dependent on MMB. By ChIP-seq and 4C-seq, the genome-wide binding of MMB upon YAP overexpression was analyzed and long-range chromatin interaction sites of selected MMB target gene promoters were identified. Strikingly, YAP strongly promoted chromatin-association of B-MYB through binding to distal enhancer elements that interact with MMB-regulated promoters through chromatin looping. Together, the findings of this thesis provide a so far unknown molecular mechanism by which YAP and MMB cooperate to regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{Fackler2014,
  author    = {Fackler, Marc},
  title     = {Biochemical characterization of GAS2L3, a target gene of the DREAM complex},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-103394},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {GAS2L3 was identified recently as a target gene of the DREAM complex (Reichert et al., 2010; Wolter et al., 2012). It was shown that GAS2L3 is expressed in a cell cycle specific manner and that depletion of the protein leads to defects in cytokinesis and genomic instability (Wolter et al., 2012). Major aim of this thesis was, to further characterize the biochemical properties and physiological function of GAS2L3. By in vitro co-sedimentation and bundling assays, GAS2L3 was identified as a cytoskeleton associated protein which bundles, binds and crosslinks F-actin and MTs. GST pulldown assays and co-immunoprecipitation experiments revealed that GAS2L3 interacts in vitro and in vivo with the chromosomal passenger complex (CPC), a very important regulator of mitosis and cytokinesis, and that the interaction is mediated by the GAR domain of GAS2L3 and the C-terminal part of Borealin and the N-terminal part of Survivin. Kinase assays showed that GAS2L3 is not a substrate of the CPC but is strongly phosphorylated by CDK1 in vitro. Depletion of GAS2L3 by shRNA influenced protein stability and activity of the CPC. However pharmacological studies showed that the decreased CPC activity is not responsible for the observed cytokinesis defects upon GAS2L3 depletion. Immunofluorescence experiments revealed that GAS2L3 is localized to the constriction zone by the CPC in a GAR dependent manner and that the GAR domain is important for proper protein function. New interacting proteins of GAS2L3 were identified by stable isotope labelling by amino acids in cell culture (SILAC) in combination with tandem affinity purification and subsequent mass spectrometrical analysis. Co-immunoprecipitation experiments further confirmed the obtained mass spectrometrical data. To address the physiological function of GAS2L3 in vivo, a conditional and a non-conditional knockout mouse strain was established. The non-conditional mouse strain showed a highly increased mortality rate before weaning age probably due to heart failure. The physiological function of GAS2L3 in vivo as well as the exact reason for the observed heart phenotype is not known at the moment.},
  subject      = {Zellzyklus},
  language  = {en}
}
@phdthesis{Walz2014,
  author    = {Walz, Susanne},
  title     = {DNA-Bindung von Myc und Miz1 und transkriptionelle Regulation ihrer Zielgene},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-106249},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Die Deregulation des Transkriptionsfaktors Myc ist ein charakteristisches Merkmal f{\"u}r eine Vielzahl von humanen Tumoren. Durch die transkriptionelle Aktivierung von Genen, die im Zusammenhang mit Metabolismus, Translation und Proliferation stehen, wird dadurch das Tumorwachstum beg{\"u}nstigt. Myc bildet zudem mit dem Zinkfinger-Protein Miz1 einen Komplex, der hemmend auf die Transkription von Zielgenen wirkt. Bisher sind nur wenige Myc/Miz1-reprimierte Zielgene bekannt. In der vorliegenden Arbeit konnten genomweit die DNA-Bindestellen von Myc und Miz1 durch Chromatin-Immunpr{\"a}zipitationen gefolgt von Hochdurchsatzsequenzierung in einer Zervixkarzinomzelllinie bestimmt werden. Es konnte gezeigt werden, dass Myc an Promotoren aller drei RNA-Polymerasen sowie in enhancer-Regionen bindet, w{\"a}hrend Miz1 Kernpromotoren von RNA-Polymerase II- und III-transkribierten Genen besetzt. reChIP-Experimente zeigten, dass Myc und Miz1 als Komplex an Promotoren von Zielgenen binden. Zudem wurde ein Miz1-DNA-Bindemotiv identifiziert und der transaktivierende Einfluss von Miz1 auf Gene mit diesem Motiv nachgewiesen. Das {\"u}berwiegende Vorhandensein von Myc/Max-Komplexen f{\"u}hrt zu einer Transaktivierung von E-Box-haltigen Promotoren. Andererseits erfolgt die transkriptionelle Repression von Myc/Miz1-Zielgenen an Promotoren, an denen der Myc/Miz1-Komplex vorherrscht. In aktuellen Publikationen konnte gezeigt werden, dass nach mitogener Stimulation von Lymphozyten es zu einer Erh{\"o}hung der Myc-Expression kommt, wodurch Myc als ein genereller Transkriptionsaktivator fungiert, der alle Gene gleichermaßen induziert. Trotz hoher Myc-Mengen in Tumorzellen konnte die generelle Myc-vermittelte Transaktivierung nicht nachgewiesen werden. Zus{\"a}tzlich zur Myc-abh{\"a}ngigen Transaktivierung von E-Box-haltigen Genen, z. B. beteiligt an Translation und RNA-Prozessierung, und der Miz1-vermittelten transkriptionellen Aktivierung von Genen mit Miz1-Motiv (z. B. involviert in Autophagie), konnte entgegen dem Modell der generellen Genamplifikation durch Myc eine Myc/Miz1-abh{\"a}ngige Repression von Zielgenen belegt werden. Die neu gewonnenen Erkenntnisse des Bindeverhaltens des Myc/Miz1-Komplexes und der daraus resultierenden transkriptionellen Regulation von Myc/Miz1-Zielgenen erm{\"o}glichen ein besseres Verst{\"a}ndnis der Myc-Funktion in Tumorzellen und k{\"o}nnte zur Verbesserung von Tumortherapien f{\"u}hren.},
  subject      = {Myc},
  language  = {de}
}
@phdthesis{Wolter2015,
  author    = {Wolter, Patrick},
  title     = {Characterization of the mitotic localization and function of the novel DREAM target GAS2L3 and Mitotic kinesins are regulated by the DREAM complex, often up-regulated in cancer cells, and are potential targets for anti-cancer therapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122531},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The recently discovered human DREAM complex (for DP, RB-like, E2F and MuvB complex) is a chromatin-associated pocket protein complex involved in cell cycle- dependent gene expression. DREAM consists of five core subunits and forms a complex either with the pocket protein p130 and the transcription factor E2F4 to repress gene expression or with the transcription factors B-MYB and FOXM1 to promote gene expression. Gas2l3 was recently identified by our group as a novel DREAM target gene. Subsequent characterization in human cell lines revealed that GAS2L3 is a microtubule and F-actin cross-linking protein, expressed in G2/M, plays a role in cytokinesis, and is important for chromosomal stability. The aim of the first part of the study was to analyze how expression of GAS2L3 is regulated by DREAM and to provide a better understanding of the function of GAS2L3 in mitosis and cytokinesis. ChIP assays revealed that the repressive and the activating form of DREAM bind to the GAS2L3 promoter. RNA interference (RNAi) mediated GAS2L3 depletion demonstrated the requirement of GAS2L3 for proper cleavage furrow ingression in cytokinesis. Immunofluorescence-based localization studies showed a localization of GAS2L3 at the mitotic spindle in mitosis and at the midbody in cytokinesis. Additional experiments demonstrated that the GAS2L3 GAR domain, a putative microtubule- binding domain, is responsible for GAS2L3 localization to the constriction zones in cytokinesis suggesting a function for GAS2L3 in the abscission process. DREAM is known to promote G2/M gene expression. DREAM target genes include several mitotic kinesins and mitotic microtubule-associated proteins (mitotic MAPs). However, it is not clear to what extent DREAM regulates mitotic kinesins and MAPs, so far. Furthermore, a comprehensive study of mitotic kinesin expression in cancer cell lines is still missing. Therefore, the second major aim of the thesis was to characterize the regulation of mitotic kinesins and MAPs by DREAM, to investigate the expression of mitotic kinesins in cancer cell line panels and to evaluate them as possible anti-cancer targets. ChIP assays together with RNAi mediated DREAM subunit depletion experiments demonstrated that DREAM is a master regulator of mitotic kinesins. Furthermore, expression analyses in a panel of breast and lung cancer cell lines revealed that mitotic kinesins are up-regulated in the majority of cancer cell lines in contrast to non-transformed controls. Finally, an inducible lentiviral-based shRNA system was developed to effectively deplete mitotic kinesins. Depletion of selected mitotic kinesins resulted in cytokinesis failures and strong anti-proliferative effects in several human cancer cell lines. Thus, this system will provide a robust tool for future investigation of mitotic kinesin function in cancer cells.},
  subject      = {Zellzyklus},
  language  = {en}
}
@phdthesis{Wurster2014,
  author    = {Wurster, Sebastian},
  title     = {Die Bedeutung von LIN9 f{\"u}r die Regulation der Genexpression, die genomische Stabilit{\"a}t und die Tumorsuppression},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114967},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Pocket proteins and E2F transcription factors regulate the expression of cell cycle associated genes and play a central role in the coordination of cell division, differentiation, and apoptosis. Disorders of these pathways contribute to the development of various human tumor entities. Despite intensive research in the field of cell cycle regulation many details are not yet understood. The LIN complex (LINC / DREAM) is a recently discovered human multiprotein complex, which dynamically interacts with pocket proteins and E2F transcription factors. An essential component of the LIN complex is the LIN9 protein. In order to obtain a better insight into the function of this protein in cell cycle regulation and tumorigenesis, a conditional Lin9 knockout mouse model was established in our laboratory. The primary objective of this study was the phenotypic characterization of embryonic fibroblasts (MEFs) from these mice. Shortly after inactivation of Lin9 cell proliferation was massively impaired. Multiple types of mitotic defects such as structural abnormalities of the spindle apparatus, aberrant nuclei, failed nuclear segregation and cytokinesis failure have been observed in Lin9-depleted cells leading to a dramatic increase in polyploid and aneuploid cells. Ultimately these serious aberrations result in premature cellular senescence. If the senescence of Lin9-deficient cells is overcome by the Large T antigen the cells can adhere to the loss of Lin9, but show severe genomic instability and grow anchorage-independently in soft-agar as a sign of oncogenic transformation. In the second part of the thesis the gene expression of Lin9-deficient cells was assessed by quantitative real time PCR analyses to determine, whether the mitotic abnormalities are caused by transcriptional defects. Here a significant reduction of mitotic gene expression was observed in Lin9-depleted cells. Additionally chromatin immunoprecipitation experiments were performed to clarify the underlying molecular mechanisms. Compared to control cells epigenetic alterations at the promoters of mitotic target genes with regard to activating histone modifications were found in Lin9-deficient MEFs. In the last section of this study, the effects of Lin9 heterozygosity were analyzed. Lin9 heterozygous MEFs showed normal proliferation, although expression of different mitotic genes was slightly reduced. It appeared, however, that the mitotic spindle checkpoint of Lin9 heterozygous MEFs is weakened and thus over several cell generations an increase in polyploid cells was observed. Soft-agar assays showed that Lin9 heterozygosity contributes to oncogenic transformation. Taken together, these results document a crucial role of LIN9 in the regulation of cell cycle-associated gene expression. LIN9 is an essential factor for cell proliferation on one hand, while at the same time it functions as a tumor suppressor.},
  subject      = {Zellzyklus},
  language  = {de}
}
@phdthesis{Kumari2014,
  author    = {Kumari, Geeta},
  title     = {Molecular Characterization of the Induction of Cell Cycle Inhibitor p21 in Response to Inhibition of the Mitotic Kinase Aurora B},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-101327},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Aurora B ist eine mitotische Kinase, die entscheidende Funktionen in der Zellteilung aus{\"u}bt. Aurora B ist außerdem in einer Vielzahl von Krebsarten mutiert oder {\"u}berexprimiert. Daher ist die Aurora B Kinase ein attraktives Ziel f{\"u}r die Tumortherapie. Gegenw{\"a}rtig werden Aurora B-Inhibitoren zur Behandlung von soliden Tumoren und Leuk{\"a}mien in verschiedenen klinischen Studien getestet. Es fehlen jedoch Informationen, welche molekularen Mechanismen den beschriebenen Ph{\"a}notypen wie Zellzyklusarrest, Aktivierung des Tumorsuppressors p53 und seines Zielgens p21 nach Aurora B-Hemmung zugrunde liegen. Hauptziel dieser Arbeit war es die Mechanismen der p21-Induktion nach Hemmung von Aurora B zu untersuchen. Es konnte gezeigt werden, dass nach Hemmung von Aurora B die p38 MAPK phosphoryliert und somit aktiviert wird. Experimente mit p38-Inhbitoren belegen, dass p38 f{\"u}r die Induktion von p21 und den Zellzyklusarrest ben{\"o}tigt wird. Die Stabilisierung von p53 nach Aurora B-Inhibition und die Rekrutierung von p53 an den p21-Genpromotor erfolgen jedoch unabh{\"a}ngig vom p38-Signalweg. Stattdessen ist p38 f{\"u}r die Anreicherung der elongierenden RNA-Polymerase II in der kodierenden Region des p21-Gens und f{\"u}r die Bildung des p21 mRNA Transkripts notwendig. Diese Daten zeigen, dass p38 transkriptionelle Elongation des p21-Gens nach Aurora B Hemmung f{\"o}rdert. In weiteren Untersuchungen konnte ich zeigen, dass die Aurora B-Hemmung zu einer Dephosphorylierung des Retinoblastoma-Proteins f{\"u}hrt und dadurch eine Abnahme der E2F-abh{\"a}ngigen Transkription bewirkt. Dies l{\"o}st indirekt einen Zellzyklusarrest aus. Weiterhin konnte mit Hilfe von synchronisierten Zellen gezeigt werden, dass p21 nach Durchlaufen einer abnormalen Mitose induziert wird, jedoch nicht nach Aurora B-Hemmung in der Interphase. Interessanterweise werden p38, p53 und p21 schon bei partieller Inhibition von Aurora B aktiviert. Die partielle Inhibition von Aurora B f{\"u}hrt zu chromosomaler Instabilit{\"a}t aber nicht zum Versagen der Zytokinese und zur Bildung polyploider Zellen. Damit korreliert die Aktivierung des p38-p53-p21-Signalweges nicht mit Tetraploidie sondern mit vermehrter Aneuploidie. Die partielle Hemmung von Aurora B f{\"u}hrt außerdem zur vermehrten Entstehung von reaktive Sauerstoffspezies (ROS), welche f{\"u}r die Aktivierung von p38, p21 und f{\"u}r den Zellzyklusarrest ben{\"o}tigt werden. Basierend auf diesen Beobachtungen kann folgendes Modell postuliert werden: Die Hemmung von Aurora B f{\"u}hrt zu Fehlern in der Chromosomenverteilung in der Mitose und damit zu Aneuploidie. Dies f{\"u}hrt zu vermehrter Produktion von ROS, m{\"o}glicherweise durch proteotoxischer Stress, hervorgerufen durch die Imbalanz der Proteinbiosynthese in aneuploiden Zellen. ROS bewirkt eine Aktivierung der p38 MAPK und tr{\"a}gt damit zur Induktion von p21 und dem resultierenden Zellzyklusarrest bei. Aneuploidie, proteotoxischer und oxidativer Stress stellen Schl{\"u}sselmerkmale von Tumorkrankungen dar. Anhand der Ergebnisse dieser Arbeit k{\"o}nnte die Kombination von Aurora B-Hemmstoffen mit Medikamenten, die gezielt aneuploide Zellen angreifen, in Tumorerkrankungen therapeutisch wirksam sein.},
  subject      = {Zellzyklus},
  language  = {en}
}
@phdthesis{Esterlechner2013,
  author    = {Esterlechner, Jasmina},
  title     = {Role of the DREAM complex in mouse embryonic stem cells and identification of ZO-2 as a new LIN9 interacting protein},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-90440},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {The DREAM complex plays an important role in regulation of gene expression during the cell cycle. It was previously shown that the DREAM subunits LIN9 and B-MYB are required for early embryonic development and for the maintenance of the inner cell mass in vitro. In this work the effect of LIN9 or B-MYB depletion on embryonic stem cells (ESC) was examined. It demonstrates that LIN9 and B-MYB knock down changes the cell cycle distribution of ESCs and results in an accumulation of cells in G2 and M and in an increase of polyploid cells. By using genome-wide expression studies it was revealed that the depletion of LIN9 leads to downregulation of mitotic genes and to upregulation of differentiation-specific genes. ChIP-on chip experiments determined that mitotic genes are direct targets of LIN9 while lineage specific markers are regulated indirectly. Importantly, depletion of LIN9 does not alter the expression of the pluripotency markers Sox2 and Oct4 and LIN9 depleted ESCs retain alkaline phosphatase activity. I conclude that LIN9 is essential for proliferation and genome stability of ESCs by activating genes with important functions in mitosis and cytokinesis. The exact molecular mechanisms behind this gene activation are still unclear as no DREAM subunit features a catalytically active domain. It is assumed that DREAM interacts with other proteins or co-factors for transcriptional activation. This study discovered potential binding proteins by combining in vivo isotope labeling of proteins with mass spectrometry (MS) and further analysed the identified interaction of the tight junction protein ZO-2 with DREAM which is cell cycle dependent and strongest in S-phase. ZO-2 depletion results in reduced cell proliferation and decreased G1 gene expression. As no G2/M genes, typical DREAM targets, are affected upon ZO-2 knock down, it is unlikely that ZO-2 binding is needed for a functional DREAM complex. However, this work demonstrates that with (MS)-based quantitative proteomics, DREAM interacting proteins can be identified which might help to elucidate the mechanisms underlying DREAM mediated gene activation.},
  subject      = {Zellzyklus},
  language  = {en}
}
@phdthesis{Fuhr2023,
  author    = {Fuhr, Viktoria},
  title     = {Target Identification and Validation in Ibrutinib-treated Mantle Cell Lymphoma},
  doi       = {10.25972/OPUS-31059},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-310595},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Ibrutinib serves as an efficient second-line therapy in relapsed/refractory mantle cell lymphoma. However, resistance to the BTK inhibitor results in a poor prognosis for patients. Since the mechanisms leading to resistance in initially responding tumor cells are poorly understood, this work aimed to decipher acquired features in ibrutinib-surviving cells of a sensitive mantle cell lymphoma cell line and evaluate these potential therapeutic targets in ibrutinib-treated mantle cell lymphoma. Time-resolved single-cell RNA sequencing was performed to track the transcriptomic evolution of REC-1 cells across 6 and 48 hours of treatment. Single-cell analysis uncovered a subpopulation of REC-1 with potentially greater aggressiveness and survival advantage by benefiting from interaction with the tumor microenvironment. Upregulation of B-cell receptor genes, elevated surface antigen expression of CD52 and metabolic rewiring to higher dependence on oxidative phosphorylation were identified as further potential resistance features of ibrutinib-surviving cells. RNA sequencing after prolonged incubation corroborated the increase in CD52 and oxidative phosphorylation as dominant characteristics of the cells surviving the 4-day treatment, highlighting their potential as therapeutic targets in combination with ibrutinib treatment. Concomitant use of ibrutinib and the oxidative phosphorylation inhibitor IACS-010759 increased toxicity compared to ibrutinib monotherapy due to higher apoptosis and greater inhibition of proliferation. For anti-CD52 therapy, a consecutive approach with ibrutinib pretreatment followed by incubation of surviving cells with a CD52 monoclonal antibody and human serum yielded a synergistic effect, as ibrutinib-surviving mantle cell lymphoma cells were rapidly depleted by complement-dependent cytotoxicity. Regarding the effects on primary tumor cells from mantle cell lymphoma patients, ibrutinib induced upregulation of CD52 in some cases, and increased toxicity of anti-CD52 therapy was observed in ibrutinib-sensitive patient samples after pretreatment with the BTK inhibitor. The likely favorable in vivo efficacy of an anti-CD52 therapy might therefore be restricted to a subgroup of mantle cell lymphoma patients, also in view of the associated side effects. Given the need for new therapeutic options in mantle cell lymphoma to overcome resistance to ibrutinib, this work highlights the potentially beneficial use of an oxidative phosphorylation inhibitor as add-on therapy. In addition, the findings suggest to further assess the value of anti-CD52 therapy as consolidation to ibrutinib in ibrutinib-sensitive patients with elevated CD52 surface levels on tumor cells to target resistant clones and minimize risk of minimal residual disease and relapse.},
  subject      = {B-Zell-Lymphom},
  language  = {en}
}
@phdthesis{Hanselmann2023,
  author    = {Hanselmann, Steffen},
  title     = {PRC1 serves as a microtubule-bundling protein and is a potential therapeutic target for lung cancer},
  doi       = {10.25972/OPUS-26631},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-266314},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Protein regulator of cytokinesis 1 (PRC1) is a microtubule-associated protein with essential roles in mitosis and cytokinesis. Furthermore, the protein is highly expressed in several cancer types which is correlated with aneuploidy and worse patient outcome. In this study it was investigated, whether PRC1 is a potential target for lung cancer as well as its possible nuclear role. Elevated PRC1 expression was cell cycle-dependent with increasing levels from S-phase to G2/M-phase of the cell cycle. Thereby, PRC1 localized at the nucleus during interphase and at the central spindle and midbody during mitosis and cytokinesis. Genome-wide expression profiling by RNA sequencing of ectopically expressed PRC1 resulted in activation of the p53 pathway. A mutant version of PRC1, that is unable to enter the nucleus, induced the same gene sets as wildtype PRC1, suggesting that PRC1 has no nuclear-specific functions in lung cancer cells. Finally, PRC1 overexpression leads to proliferation defects, multi-nucleation, and enlargement of cells which was directly linked to microtubule-bundling within the cytoplasm. For analysis of the requirement of PRC1 in lung cancer, different inducible cell lines were generated to deplete the protein by RNA interference (RNAi) in vitro. PRC1 depletion caused proliferation defects and cytokinesis failures with increased numbers of bi- and multi-nucleated cells compared to non-induced lung cancer cells. Importantly, effects in control cells were less severe as in lung cancer cells. Finally, p53 wildtype lung cancer cells became senescent, whereas p53 mutant cells became apoptotic upon PRC1 depletion. PRC1 is also required for tumorigenesis in vivo, which was shown by using a mouse model for non-small cell lung cancer driven by oncogenic K-RAS and loss of p53. Here, lung tumor area, tumor number, and high-grade tumors were significantly reduced in PRC1 depleted conditions by RNAi. In this study, it is shown that PRC1 serves as a microtubule-bundling protein with essential roles in mitosis and cytokinesis. Expression of the protein needs to be tightly regulated to allow unperturbed proliferation of lung cancer cells. It is suggested that besides phosphorylation of PRC1, the nuclear localization might be a protective mechanism for the cells to prevent perinuclear microtubule-bundling. In conclusion, PRC1 could be a potential target of lung cancer as mono therapy or in combination with a chemotherapeutic agent, like cisplatin, which enhanced the negative effects on proliferation of lung cancer cells in vitro.},
  language  = {en}
}
@phdthesis{Engelmann2023,
  author    = {Engelmann, Daria Marie},
  title     = {Regulation of Mammalian Phosphoglycolate Phosphatase},
  doi       = {10.25972/OPUS-19957},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199577},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Mammalian phoshoglycolate phosphatase (PGP, also known as AUM) belongs to the ubiquitous HAD superfamily of phosphatases. As several other members of HAD phosphatases, the Mg2+-dependent dephosphorylation is conducted via a nucleophilic attack from a conserved aspartate residue in the catalytic cleft. The protein structure of PGP could not yet be solved entirely. Only a hybrid consisting of the PGP cap and the PDXP core (pyridoxal phosphatase, closest enzyme paralog) was crystallizable so far. PGP is able to efficiently dephosphorylate 2-phosphoglycolate, 2-phospho-L-lactate, 4-phospho-D-erythronate, and glycerol-3-phosphate in vitro which makes them likely physiological substrates. The first three substrates can be derived from metabolic side reactions (during glycolysis) and inhibit key enzymes in glycolysis and pentose phosphate pathway, the latter is situated at the intersection between glycolysis and lipogenesis. 2-phosphoglycolate can also be released in the context of repair of oxidative DNA damage. The activity of purified PGP can be reversibly inhibited by oxidation - physiologically likely in association with epidermal growth factor (EGF) signal transduction. In fact, an association between persistently lacking PGP activity (via downregulation) and the presence of hyperphosphorylated proteins after EGF stimulation has been identified. Reversible oxidation and transient inactivation of PGP may be particularly important for short-term and feedback regulatory mechanisms (as part of the EGF signaling). Furthermore, cellular proliferation in PGP downregulated cells is constantly reduced. Whole-body PGP inactivation in mice is embryonically lethal. Despite the many well-known features and functions, the knowledge about PGP is still incomplete. In the present work the influence of reactive oxygen species (ROS) on PGP activity in cells und a possible connection between oxidative stress and the proliferation deficit of PGP downregulated cells was investigated. For the experiments, a spermatogonial cell line was used (due to the high PGP expression in testis). PGP activity can be reversibly inhibited in cellular lysates by H2O2 (as a ROS representative). Reversible oxidation could thus indeed be physiologically important. More oxidative DNA damage (by bleomycin) showed no PGP-dependent effects here. EGF stimulation (as an inducer of transient and well-controlled ROS production), low concentrations of menadione (as an oxidant) and N-acetylcysteine (as an antioxidant) were able to approximate the proliferation rate in PGP downregulated cells to that of control cells. The redox regulation of PGP could thus have an influence on cellular proliferation as a feedback mechanism - a mechanism that could not take place in PGP downregulated cells. However, the connections are probably even more complex and cannot be elucidated by a sole examination of the proliferation rate. The present results can thus only be regarded as preliminary experiments. For a better understanding of the features and functions of PGP, this work then focused on specific regulation of enzyme activity by pharmacologically applicable small molecules. Four potent inhibitors had previously been identified in a screening campaign. In this work, three of these four inhibiting compounds could be further characterized in experiments with highly purified, recombinant murine and human PGP. Compounds \#2 and \#9 showed competitive inhibition properties with a markedly rising KM value with little or no change in vmax. The results were consistent for all tested protein variants: the murine and the human PGP as well as a PGP/PDXP hybrid protein. Compound \#1 was the most potent and interesting PGP-inhibitory molecule: less change in KM and a constant decrease in vmax as well as a lower impact on the PGP/PDXP hybrid hint at a mixed mode of inhibition as a combination of competitive and non-competitive inhibition. The characterization of the potential inhibitors can serve as a basis for further structural analysis and studies on the complex physiological role of PGP.},
  subject      = {Phosphoglykolatphosphatase},
  language  = {en}
}
@phdthesis{JimenezMartin2022,
  author    = {Jim{\´e}nez Mart{\´i}n, Ovidio Manuel},
  title     = {Analysis of MYCN and MAX alterations in Wilms Tumor},
  doi       = {10.25972/OPUS-24291},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-242919},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Wilms tumor (WT) is the most common renal tumor in childhood. Among others, MYCN copy number gain and MYCN P44L and MAX R60Q mutations have been identified in WT. The proto-oncogene MYCN encodes a transcription factor that requires dimerization with MAX to activate transcription of numerous target genes. MYCN gain has been associated with adverse prognosis. The MYCN P44L and MAX R60Q mutations, located in either the transactivating or basic helix-loop-helix domain, respectively, are predicted to be damaging by different pathogenicity prediction tools. These mutations have been reported in several other cancers and remain to be functionally characterized. In order to further describe these events in WT, we screened both mutations in a large cohort of unselected WT patients, to check for an association of the mutation status with certain histological or clinical features. MYCN P44L and MAX R60Q revealed frequencies of 3 \% and 0.9 \% and also were significantly associated to higher risk of relapse and metastasis, respectively. Furthermore, to get a better understanding of the MAX mutational landscape in WT, over 100 WT cases were analyzed by Sanger sequencing to identify other eventual MAX alterations in its coding sequence. R60Q remained the only MAX CDS alteration described in WT to date. To analyze the potential functional consequences of these mutations, we used a doxycycline-inducible system to overexpress each mutant in HEK293 cells. This biochemical characterization identified a reduced transcriptional activation potential for MAX R60Q, while the MYCN P44L mutation did not change activation potential or protein stability. The protein interactome of N-MYC-P44L was likewise not altered as shown by mass spectrometric analyses of purified N-MYC complexes. However, we could identify a number of novel N-MYC partner proteins, several of these known for their oncogenic potential. Their correlated expression in WT samples suggested a role in WT oncogenesis and they expand the range of potential biomarkers for WT stratification and targeting, especially for high-risk WT.},
  subject      = {Nephroblastom},
  language  = {en}
}
@phdthesis{PrietoGarcia2022,
  author    = {Prieto Garc{\´i}a, Cristian},
  title     = {USP28 regulates Squamous cell oncogenesis and DNA repair via ΔNp63 deubiquitination},
  doi       = {10.25972/OPUS-27033},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270332},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {∆Np63 is a master regulator of squamous cell identity and regulates several signaling pathways that crucially contribute to the development of squamous cell carcinoma (SCC) tumors. Its contribution to coordinating the expression of genes involved in oncogenesis, epithelial identity, DNA repair, and genome stability has been extensively studied and characterized. For SCC, the expression of ∆Np63 is an essential requirement to maintain the malignant phenotype. Additionally, ∆Np63 functionally contributes to the development of cancer resistance toward therapies inducing DNA damage. SCC patients are currently treated with the same conventional Cisplatin therapy as they would have been treated 30 years ago. In contrast to patients with other tumor entities, the survival of SCC patients is limited, and the efficacy of the current therapies is rather low. Considering the rising incidences of these tumor entities, the development of novel SCC therapies is urgently required. Targeting ∆Np63, the transcription factor, is a potential alternative to improve the therapeutic response and clinical outcomes of SCC patients. However, ∆Np63 is considered "undruggable." As is commonly observed in transcription factors, ∆Np63 does not provide any suitable domains for the binding of small molecule inhibitors. ∆Np63 regulates a plethora of different pathways and cellular processes, making it difficult to counteract its function by targeting downstream effectors. As ∆Np63 is strongly regulated by the ubiquitin-proteasome system (UPS), the development of deubiquitinating enzyme inhibitors has emerged as a promising therapeutic strategy to target ∆Np63 in SCC treatment. This work involved identifying the first deubiquitinating enzyme that regulates ∆Np63 protein stability. Stateof-the-art SCC models were used to prove that USP28 deubiquitinates ∆Np63, regulates its protein stability, and affects squamous transcriptional profiles in vivo and ex vivo. Accordingly, SCC depends on USP28 to maintain essential levels of ∆Np63 protein abundance in tumor formation and maintenance. For the first time, ∆Np63, the transcription factor, was targeted in vivo using a small molecule inhibitor targeting the activity of USP28. The pharmacological inhibition of USP28 was sufficient to hinder the growth of SCC tumors in preclinical mouse models. Finally, this work demonstrated that the combination of Cisplatin with USP28 inhibitors as a novel therapeutic alternative could expand the limited available portfolio of SCC therapeutics. Collectively, the data presented within this dissertation demonstrates that the inhibition of USP28 in SCC decreases ∆Np63 protein abundance, thus downregulating the Fanconi anemia (FA) pathway and recombinational DNA repair. Accordingly, USP28 inhibition reduces the DNA damage response, thereby sensitizing SCC tumors to DNA damage therapies, such as Cisplatin.},
  language  = {en}
}
@phdthesis{Iltzsche2017,
  author    = {Iltzsche, Fabian},
  title     = {The Role of DREAM/MMB-mediated mitotic gene expression downstream of mutated K-Ras in lung cancer},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154108},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The evolutionary conserved Myb-MuvB (MMB) multiprotein complex has an essential role in transcriptional activation of mitotic genes. MMB target genes as well as the MMB associated transcription factor B-Myb and FoxM1 are highly expressed in a range of different cancer types. The elevated expression of these genes correlates with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. Although MMB has been extensively characterized biochemically, the requirement for MMB to tumorigenesis in vivo remains largely unknown and has not been tested directly so far. In this study, conditional knockout of the MMB core member Lin9 inhibits tumor formation in vivo in a mouse model of lung cancer driven by oncogenic K-Ras and loss of p53. The incomplete recombination observed within tumors points towards an enormous selection pressure against the complete loss of Lin9. RNA interference (RNAi)-mediated depletion of Lin9 or the MMB associated subunit B-Myb provides evidence that MMB is required for the expression of mitotic genes in lung cancer cells. Moreover, it was demonstrated that proliferation of lung cancer cells strongly depends on MMB. Furthermore, in this study, the relationship of MMB to the p53 tumor suppressor was investigated in a primary lung cancer cell line with restorable p53 function. Expression analysis revealed that mitotic genes are downregulated after p53 re-expression. Moreover, activation of p53 induces formation of the repressive DREAM complex and results in enrichment of DREAM at mitotic gene promoters. Conversely, MMB is displaced at these promoters. Based on these findings the following model is proposed: In p53-negative cells, mitogenic stimuli foster the switch from DREAM to MMB. Thus, mitotic genes are overexpressed and may promote chromosomal instability and tumorigenesis. This study provides evidence that MMB contributes to the upregulation of G2/M phase-specific genes in p53-negative cells and suggests that inhibition of MMB (or its target genes) might be a strategy for treatment of lung cancer.},
  subject      = {Nicht-kleinzelliges Bronchialkarzinom (NSCLC)},
  language  = {en}
}
@phdthesis{Kalb2021,
  author    = {Kalb, Jacqueline},
  title     = {The role of BRCA1 and DCP1A in the coordination of transcription and replication in neuroblastoma},
  doi       = {10.25972/OPUS-24871},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248711},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The deregulation of the MYC oncoprotein family plays a major role in tumorigenesis and tumour maintenance of many human tumours. Because of their structure and nuclear localisation, they are defined as undruggable targets which makes it difficult to find direct therapeutic approaches. An alternative approach for targeting MYC-driven tumours is the identification and targeting of partner proteins which score as essential in a synthetic lethality screen. Neuroblastoma, an aggressive entity of MYCN-driven tumours coming along with a bad prognosis, are dependent on the tumour suppressor protein BRCA1 as synthetic lethal data showed. BRCA1 is recruited to promoter regions in a MYCN-dependent manner. The aim of this study was to characterise the role of BRCA1 in neuroblastoma with molecular biological methods. BRCA1 prevents the accumulation of RNA Polymerase II (RNAPII) at the promoter region. Its absence results in the formation of DNA/RNA-hybrids, so called R-loops, and DNA damage. To prevent the accumulation of RNAPII, the cell uses DCP1A, a decapping factor known for its cytoplasmatic and nuclear role in mRNA decay. It is the priming factor in the removal of the protective 5'CAP of mRNA, which leads to degradation by exonucleases. BRCA1 is necessary for the chromatin recruitment of DCP1A and its proximity to RNAPII. Cells showed upon acute activation of MYCN a higher dependency on DCP1A. Its activity prevents the deregulation of transcription and leads to proper coordination of transcription and replication. The deregulation of transcription in the absence of DCP1A results in replication fork stalling and leads to activation of the Ataxia telangiectasia and Rad3 related (ATR) kinase. The result is a disturbed cell proliferation to the point of increased apoptosis. The activation of the ATR kinase pathway in the situation where DCP1A is knocked down and MYCN is activated, makes those cells more vulnerable for the treatment with ATR inhibitors. In summary, the tumour suppressor protein BRCA1 and the decapping factor DCP1A, mainly known for its function in the cytoplasm, have a new nuclear role in a MYCN-dependent context. This study shows their essentiality in the coordination of transcription and replication which leads to an unrestrained growth of tumour cells if uncontrolled.},
  subject      = {Neuroblastom},
  language  = {en}
}
@phdthesis{FetivaMora2023,
  author    = {Fetiva Mora, Maria Camila},
  title     = {Changes in chromatin accessibility by oncogenic YAP and its relevance for regulation of cell cycle gene expression and cell migration},
  doi       = {10.25972/OPUS-30291},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-302910},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Various types of cancer involve aberrant cell cycle regulation. Among the pathways responsible for tumor growth, the YAP oncogene, a key downstream effector of the Hippo pathway, is responsible for oncogenic processes including cell proliferation, and metastasis by controlling the expression of cell cycle genes. In turn, the MMB multiprotein complex (which is formed when B-MYB binds to the MuvB core) is a master regulator of mitotic gene expression, which has also been associated with cancer. Previously, our laboratory identified a novel crosstalk between the MMB-complex and YAP. By binding to enhancers of MMB target genes and promoting B-MYB binding to promoters, YAP and MMB co-regulate a set of mitotic and cytokinetic target genes which promote cell proliferation. This doctoral thesis addresses the mechanisms of YAP and MMB mediated transcription, and it characterizes the role of YAP regulated enhancers in transcription of cell cycle genes. The results reported in this thesis indicate that expression of constitutively active, oncogenic YAP5SA leads to widespread changes in chromatin accessibility in untransformed human MCF10A cells. ATAC-seq identified that newly accessible and active regions include YAP-bound enhancers, while the MMB-bound promoters were found to be already accessible and remain open during YAP induction. By means of CRISPR-interference (CRISPRi) and chromatin immuniprecipitation (ChIP), we identified a role of YAP-bound enhancers in recruitment of CDK7 to MMB-regulated promoters and in RNA Pol II driven transcriptional initiation and elongation of G2/M genes. Moreover, by interfering with the YAP-B-MYB protein interaction, we can show that binding of YAP to B-MYB is also critical for the initiation of transcription at MMB-regulated genes. Unexpectedly, overexpression of YAP5SA also leads to less accessible chromatin regions or chromatin closing. Motif analysis revealed that the newly closed regions contain binding motifs for the p53 family of transcription factors. Interestingly, chromatin closing by YAP is linked to the reduced expression and loss of chromatin-binding of the p53 family member Np63. Furthermore, I demonstrate that downregulation of Np63 following expression of YAP is a key step in driving cellular migration. Together, the findings of this thesis provide insights into the role of YAP in the chromatin changes that contribute to the oncogenic activities of YAP. The overexpression of YAP5SA not only leads to the opening of chromatin at YAP-bound enhancers which together with the MMB complex stimulate the expression of G2/M genes, but also promotes the closing of chromatin at ∆Np63 -bound regions in order to lead to cell migration.},
  subject      = {Chromatin},
  language  = {en}
}