Cancer is one of the leading causes of death worldwide. The underlying tumorigenesis is driven by the accumulation of alterations in the genome, eventually disabling tumor suppressors and activating proto-oncogenes. The MYC family of proto-oncogenes shows a strong deregulation in the majority of tumor entities. However, the exact mechanisms that contribute to MYC-driven oncogenesis remain largely unknown. Over the past decades, the influence of the MYC protein on transcription became increasingly apparent and was thoroughly investigated. Additionally, in recent years several publications provided evidence for so far unreported functions of MYC that are independent of a mere regulation of target genes. These findings suggest an additional role of MYC in the maintenance of genomic stability and this role is strengthened by key findings presented in this thesis. In the first part, I present data revealing a pathway that allows MYC to couple transcription elongation and DNA double-strand break repair, preventing genomic instability of MYC-driven tumor cells. This pathway is driven by a rapid transfer of the PAF1 complex from MYC onto RNAPII, a process that is mediated by HUWE1. The transfer controls MYC-dependent transcription elongation and, simultaneously, the remodeling of chromatin structure by ubiquitylation of histone H2B. These regions of open chromatin favor not only elongation but also DNA double-strand break repair. In the second part, I analyze the ability of MYC proteins to form multimeric structures in response to perturbation of transcription and replication. The process of multimerization is also referred to as phase transition. The observed multimeric structures are located proximal to stalled replication forks and recruit factors of the DNA-damage response and transcription termination machinery. Further, I identified the HUWE1-dependent ubiquitylation of MYC as an essential step in this phase transition. Cells lacking the ability to form multimers display genomic instability and ultimately undergo apoptosis in response to replication stress. Both mechanisms present MYC as a stress resilience factor under conditions that are characterized by a high level of transcriptional and replicational stress. This increased resilience ensures oncogenic proliferation. Therefore, targeting MYC’s ability to limit genomic instability by uncoupling transcription elongation and DNA repair or disrupting its ability to multimerize presents a therapeutic window in MYC-dependent tumors. / Tumorerkrankungen sind eine der häufigsten Todesursachen weltweit. Für die Entstehung und Entwicklung eines Tumors sind Veränderungen im Genom verantwortlich, wobei Proto-Onkogene aktiviert und Tumorsuppressorgene inaktiviert werden. Die MYC-Familie der Proto-Onkogene ist in der Mehrzahl der menschlichen Tumorerkrankungen stark dereguliert. Der genaue Mechanismus, der in MYC-getriebenen Tumoren eine Rolle spielt, ist aber weiterhin ungeklärt. In den letzten Jahrzehnten wurde die Funktion von MYC als Transkriptionsfaktor in den Vordergrund gestellt. Veröffentlichungen der letzten Jahre deuten zusätzlich auf mehrere, bisher unbekannte Funktionen hin, die unabhängig von einer bloßen Regulation von Zielgenen sind und auf eine zusätzliche Rolle bei der Erhaltung der genomischen Stabilität hinweisen. Diese Rolle wird durch wesentliche Ergebnisse dieser Doktorarbeit gestärkt. In dem ersten Teil der Doktorarbeit präsentiere ich einen Pathway, der es MYC ermöglicht, transkriptionelle Elongation und Doppelstrangbruch-Reparatur zu koppeln, wodurch genomische Instabilität in MYC-gesteuerten Tumorzellen limitiert wird. Dieser Pathway wird durch einen schnellen Transfer des PAF1-Komplexes von MYC auf die RNAPII angetrieben, bei dem HUWE1 eine essenzielle Rolle einnimmt. Der Transfer steuert die MYC-abhängige transkriptionelle Elongation und gleichzeitig die Öffnung der Chromatinstruktur. Dies geschieht durch Ubiquitylierung des Histons H2B zugunsten von sowohl transkriptioneller Elongation als auch der DNA-Doppelstrangbruchreparatur. In dem zweiten Teil der Doktorarbeit analysiere ich die Fähigkeit von MYC-Proteinen, als Reaktion auf eine Störung der Transkription und/oder Replikation multimere Strukturen bilden zu können. Diese Fähigkeit wird auch als Phasentrennung bezeichnet. Die multimere Strukturen befinden sich in der Nähe von blockierten Replikationsgabeln und rekrutieren Faktoren der DNA-Schadensreaktion und der Transkriptionsterminationsmaschinerie. Die HUWE1-abhängige Ubiquitylierung von MYC habe ich als wesentlichen Schritt der Phasentrennung identifiziert. Zellen ohne die Fähigkeit zur Bildung von Multimeren zeigen als Reaktion auf Replikationsstress exzessive genomische Instabilität und letztendlich Apoptose auf. Beide Mechanismen machen MYC zu einem Faktor, der genomische Instabilität als Resultat von unphysiologischem Transkriptions- und Replikationsstress limitiert und damit die onkogene Zellteilung gewährleistet. Eine gezielte Beeinflussung der aufgeführten Mechanismen, durch welche MYC die genomische Instabilität limitiert, kann bei MYC-abhängigen Tumoren von großem therapeutischem Nutzen sein.

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Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin’s B- cell lymphoma, accounting for about 30% of these lymphomas in the United States. Large-scale genome analyses of DLBCL have identified mutations in the related histone acetyltransferases (HATs) p300 and CBP in approximately 15% of patient samples and patient-derived cell lines. The research presented herein characterizes two human DLBCL cell lines, RC-K8 and SUDHL2, which express C-terminally truncated HAT domain-deficient p300 proteins, p300ΔC-1087 and p300 p300ΔC-820, respectively. It is shown that p300ΔC-820 localizes to sites of active transcription in the nucleus, interacts with NF-κB transcription factor REL, weakly enhances REL-dependent transactivation, and has a half-life similar to wild-type p300. Results demonstrate that knockdown of p300ΔC-820 in SUDHL2 cells reduces cell proliferation in vitro. In RC-K8 cells, p300ΔC-1087 suppresses expression of the NF-κB target genes A20 and IκBα, both of which are cytotoxic when overexpressed in RC-K8 cells. Microarray analysis of p300ΔC1087 knockdown compared to wild-type RC-K8 cells indicated that p300ΔC-1087 suppresses an NF-κB gene expression program and activates a MYC gene expression program in RC-K8 cells. Bioinformatic analysis demonstrated that cancer cell lines— regardless of tissue type—with truncating p300 mutations have altered expression of a MYC target gene set as compared to cancer cell lines with wild-type p300/CBP. Taken together, this research indicates that p300 truncations contribute to cell growth in DLBCL by modifying the transcriptional output of two lymphoid cell-specific oncoproteins, NF- κB and MYC, to optimal levels and suggests that p300 truncating mutations similarly modify the activity of oncogenic drivers in other cancer cell types. Based on this work, p300 truncation is proposed to represent a new class of oncogenic mutation that serves to optimize the activity of context-specific oncogenic transcription factors, and it is suggested that such oncogenic mutations be termed “cancer modifying” mutations. / 2017-09-30T00:00:00Z

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No description available.

The Gadd45 family of proteins is known to play a central role as cellular stress sensors that modulate the response of mammalian cells to different stressors, including oncogenic stress. Gadd45a expression is regulated during myeloid cell differentiation, and is also induced in response to acute stimulation with cytokines, myeloablation and inflammation. The proto-oncogene C-myc plays a pivotal role in growth control, differentiation and apoptosis in hematopoietic cells. Deregulated Myc in hematopoietic cells blocks the differentiation program and prevents normal homeostatic cellular apoptosis, which alters the balance of cell populations, often participating in leukemogenesis. The status of Gadd45a expression has been shown to impact on different cancers, including breast cancer and leukemia. How the stress response gene Gadd45a modulates oncogenic stress imparted by deregulated c-Myc in myeloid cells has not been investigated. We hypothesized that Gadd45a and its interacting partner proteins can modulate specific pro-survival or pro-apoptotic signaling pathways, altering the cellular response to oncogenic myc in myeloid cells. Gadd45a may play different roles in proliferating and differentiating cells, and myeloid cells in vivo are at all stages of myeloid development. Therefore, to understand how Gadd45a status impacts on proliferating and differentiating myeloid cells, we decided to study the effect of loss of Gadd45a in myc-expressing cells that are either proliferating or stimulated to undergo differentiation. Therefore, to address this issue we utilized bone marrow from wild-type (wt) and Gadd45a null mice, and retrovirally infected these cells to express constitutive Myc or empty vector control. Using these cells we have shown that bone marrow deficient in Gadd45a and expressing constitutive Myc, display decreased apoptosis under proliferating conditions, yet increased apoptosis in media containing the differentiation inducing cytokine GM-CSF. We show that in expansion media loss of Gadd45a in the presence of Myc elicits its function through the activation of p38, with evidence supporting a role for PU.1 and Mcl-1 expression, which are downstream of p-p38. In contrast, deregulated C-Myc and loss of Gadd45a does not signal through p-38 in GM-CSF, but surprisingly there is a decrease in cytokine receptor expression. This data demonstrates that Gadd45a may be required for optimal cytokine receptor expression in myeloid cells, which can impact on survival of the cells. Although in primary bone marrow Gadd45a status had no effect on differentiation of Myc expressing cells, the loss of Gadd45a in Hoxb8 generated cell lines shifted differentiation towards increased neutrophils. Determining the role of Gadd45a on the biological outcome of myeloid cells in response to deregulated c-Myc will provide vital information in understanding the function of Gadd45a in the development and progression of Myc expressing myeloid leukemia. / Molecular Biology and Genetics

The peptidyl prolyl cis/trans isomerase, PPIase, has been the focus of numerous studies in the field of cell cycle regulation since proline-directed phosphorylation is an essential signaling mechanism that might arrest cancer proliferation. Pin1 is the first phosphorylation-dependent PPIase enzyme to be discovered. The Pin1 regulatory mechanism, acting on other mitotic proteins in vivo and in vitro, remains largely unknown. For the study of Pin1 function, two types of assays were used to identity ligands for Pin1: (1) The Enzyme-Linked Enzyme Binding Assay (ELEBA) for the identification of WW domain ligands, (2) a catalytic assay to identified inhibitors of Pin1 catalytic activity. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain from chemical libraries. By using the ELEBA, a pSer-Pro peptidomimetic library of 315 ligands was screened, identifying three promising ligands cis-D2, O2, and M18. Competitive Kd values for cis-D2, O2, and M18 were determined to be 263 ± 6.4, 206 ± 3.4, and 130 ± 3.0μM, respectively. Furthermore, we screened the pSer-Pro peptidomimetic library using a Pin1 discontinuous-catalytic assay to identify inhibitors of Pin1. Ligands D20 and K7 were identified to decrease more than 90% of the Pin1 catalytic activity. To investigate the nature of the Pin1 interaction with c-Myc, we synthesized and characterized four peptides corresponding to the c-Myc sequence. These peptides were used in NMR isomerization studies of Pin1 by our collaborator Dr. Jeffry Peng (University of Notre Dame). Preliminary work shows that Pin1 binds and isomerizes the Ac–LLPpTPPLSPS–NH₂ peptide at the cMyc pThr58 position. Finally, we measured a secondary kinetic isotope effect (2º KIE) to study the Pin1 catalytic mechanism of proline isomerization. The ratio of kH/kD for unlabeled and [d₃]Ser-labeled substrate gave a SKIE value of 1.34 ± 0.01. The normal 2º KIE value indicates that carbonyl-serine hybridization is not changing from sp² to sp³. This result supports substrate analogue inhibitor studies, and previous solvent and SKIE results on Pin1, that suggest a twisted amide mechanism assisted by a transient hydrogen bond in the transition state. / Ph. D.

Colorectal cancer (CRC) is the second most common tumour disease in Germany, with the sequential accumulation of certain mutations playing a decisive role in the transition from adenoma to carcinoma. In particular, deregulation of the Wnt signalling pathway and the associated deregulated expression of the MYC oncoprotein play a crucial role. Targeting MYC thus represents an important therapeutic approach in the treatment of tumours. Since direct inhibition of MYC is challenging, various approaches have been pursued to date to target MYC indirectly. The MYC 5' UTR contains an internal ribosomal entry site (IRES), which has a particular role in the initiation of MYC translation, especially in multiple myeloma. As basis for this work, it was hypothesised on the basis of previous data that translation of MYC potentially occurs via its IRES in CRC as well. Based on this, two IRES inhibitors were tested for their potential to regulate MYC expression in CRC cells. In addition, alternative, 5’ UTR-dependent translation of MYC and interacting factors were investigated. EIF3D was identified as a MYC 5' UTR binding protein which has the potential to regulate MYC expression in CRC. The results of this work suggest that there is a link between eIF3D and MYC expression/translation, rendering eIF3D a potential therapeutic target for MYC-driven CRCs. / Das kolorektale Karzinom (KRK) ist die zweithäufigste Tumorerkrankung in Deutschland, wobei die sequenzielle Akkumulation bestimmter Mutationen eine entscheidende Rolle beim Übergang vom Adenom zum Karzinom spielt. Insbesondere die Deregulation des Wnt-Signalweges und die damit verbundene deregulierte Expression des MYC-Onkoproteins spielen eine entscheidende Rolle. MYC ist ein zentraler Vermittler von Zellfunktionen und reguliert als Transkriptionsfaktor die Expression fast aller Gene sowie verschiedener RNA-Spezies. Selbst kleine Veränderungen der zellulären MYC-Konzentration können das Proliferationsverhalten beeinflussen und die Entstehung und das Fortschreiten von Tumoren fördern. Die gezielte Beeinflussung von MYC stellt daher einen wichtigen therapeutischen Ansatz für die Behandlung von Tumoren dar. Da eine direkte Hemmung von MYC aufgrund seiner Struktur herausfordernd ist, wurden bisher verschiedene Ansätze verfolgt, um MYC indirekt zu beeinflussen, etwa über seinen Interaktionspartner MAX oder auf Ebene der Stabilität, Transkription oder Translation. In unserer eigenen Forschungsgruppe lag der Schwerpunkt in den letzten Jahren speziell auf der Translation von MYC im KRK. Es konnte gezeigt werden, dass die Hemmung der kanonischen cap-abhängigen Translation nicht wie erwartet zu einer Verringerung der zellulären MYC-Level führt, was auf einen alternativen Mechanismus der MYC-Translation hindeutet, der unabhängig vom eIF4F-Komplex abläuft. Die 5'-UTR von MYC enthält eine interne ribosomale Eintrittsstelle (IRES), die eine besondere Rolle bei der Initiierung der MYC-Translation spielt, insbesondere im Multiplen Myelom. Als Grundlage für diese Arbeit wurde daher die Hypothese aufgestellt, dass die Translation von MYC im KRK möglicherweise ebenfalls über die IRES erfolgt. Auf dieser Grundlage wurden zunächst zwei publizierte IRES-Inhibitoren auf ihr Potenzial zur Regulierung der MYC-Expression in KRK-Zellen getestet. J007-IRES hatte keine Auswirkungen auf die MYC-Proteinmenge, und Cymarin scheint weitaus globalere Auswirkungen zu haben, die nicht ausschließlich auf die Verringerung der MYC-Proteinmenge zurückzuführen sind. Daher wurde weiter untersucht, inwieweit die alternative Translation von MYC generell von der 5'-UTR und damit interagierenden Faktoren abhängig ist. EIF3D wurde als MYC-5'-UTR-Bindungsprotein identifiziert, dessen Knockdown zu reduzierten MYC-Leveln, einem Proliferationsdefizit sowie einer Verringerung der globalen Proteinsynthese in KRK-Zellen führte. Darüber hinaus führte die Depletion von EIF3D zu ähnlichen Veränderungen im zellulären Genexpressionsmuster wie die Depletion von MYC, wobei viele tumorassoziierte Signalwege betroffen waren. Mittels eCLIP-seq wurde die Bindung von eIF3D an die MYC mRNA nachgewiesen, der genaue Mechanismus einer möglicherweise durch eIF3D vermittelten Translation von MYC muss jedoch weiter untersucht werden. Die Ergebnisse dieser Arbeit deuten darauf hin, dass eine Verbindung zwischen eIF3D und der MYC-Expression/Translation besteht, wodurch eIF3D zu einem potenziellen therapeutischen Ziel für MYC-getriebene KRKs wird.

Chez les métazoaires, la différenciation terminale est généralement accompagnée par une sortie définitive du cycle cellulaire. Cependant, les macrophages et très peu d'autres types cellulaires rompent avec ce dogme. En effet, il est maintenant admis que les macrophages conservent la capacité de s'auto-renouveler indépendamment des cellules souches ou progénitrices. À cet égard, nous avons démontré que la double déficience en facteurs Maf dans les macrophages (Maf-DKO) leur confère la capacité de s'auto-renouveler indéfiniment en culture sans se dé-différencier ou devenir tumorigènes. Ce phénotype d'auto-renouvellement semble être médié par un réseau transcriptionnel de de gènes régissant l'auto-renouvellement qui sont également actifs dans les cellules souches embryonnaires, parmi lesquels Myc et Klf4. Ces deux facteurs sont activés et nécessaires pour l'auto-renouvellement des Maf-DKO. De façon intéressante, l'expression de Myc seul induit une prolifération illimitée des macrophages, mais provoque une transformation tumorale. Nous avons donc cherché à décrypter les mécanismes grâce auxquels Myc et Klf4 induisent l'auto-renouvellement des macrophages, en comparaison à la transformation cellulaire causée par l'expression de Myc uniquement. En outre, nous nous sommes concentrés sur l'identification de gènes candidats permettant un auto-renouvellement illimité des macrophages, tout en les protégeant de la transformation cancéreuse. Notre objectif est de contribuer à l'identification du programme transcriptionnel régulant l'auto-renouvellement non tumoral des macrophages. / In metazoan, terminal differentiation is generally accompanied by permanent exit from the cell cycle. Yet, macrophages and very few other examples break with this dogma. Indeed, it has become evident that macrophages retain the ability to self-renew independently of stem or progenitor cells. In this regard, we have previously shown that MafB/c-Maf double deficient (Maf-DKO) macrophages are able to self-renew indefinitely in vitro without dedifferentiating or becoming tumorigenic. This self-renewal phenotype appears to be mediated by a transcriptional network of self-renewal genes also active in embryonic stem cells, among which Myc and Klf4. Interestingly, these two factors are activated and required for Maf-DKO self-renewal. By contrast, Myc alone induces an unlimited proliferation of macrophages but causes malignant transformation. We aimed to decipher the mechanisms by which Myc and Klf4 induce stem cell-like self-renewal in macrophages, in comparison to cellular transformation caused by the expression of Myc alone. Additionally, we focused on identifying candidate genes allowing an unlimited self-renewal of macrophages while protecting them from tumorigenic transformation or aberrant proliferation. Our objective is to contribute to the identification of the transcriptional program regulating non-tumorigenic self-renewal in macrophages.

As neoplasias de cabeça e pescoço constituem um importante problema de saúde pública devido à alta incidência e alguns tipos estão associados a fatores comportamentais como consumo de álcool e tabaco. Apesar desses dados, a doença, especialmente em sua fase inicial, pode ser curada e alguns tipos podem ser prevenidos. Portanto, existe a necessidade de identificar e validar novos biomarcadores em câncer de cabeça e pescoço com aplicação em prognóstico e seleção de terapias mais adequadas. Neste sentido, o objetivo deste trabalho foi validar o perfil de três proteínas, SET, hnRNP K e MARK3 em tumores de cabeça e pescoço, e verificar a potencial aplicação como marcadores de diagnóstico e prognóstico em HNSCC, bem como propor um papel para estas proteínas na tumorigênese. Foram analisadas 22 amostras de tumores de cabeça e pescoço por western blotting (WB) e 96 amostras (91 tumores, 4 biópsias e 1 controle) dispostas em duplicata em lâmina de tissue microarray, obtidas no Brasil e cedidas pelo Grupo GENCAPO, por imunohistoquímica (IHC). Os dados obtidos foram correlacionados com todos os parâmetros clínicos e patológicos e com prognóstico do paciente com HNSCC por um período de 48 meses. Os resultados obtidos por WB e IHC mostraram acúmulo e fragmentação da SET e acúmulo nuclear e citoplasmático da hnRNP K nos tumores comparado a respectiva margem cirúrgica e tecido normal. A hnRNPK mostrou valor prognóstico sendo associada a sobrevida global do paciente. A proteína c-Myc e a sua forma fosforilada foram analisadas nas amostras de tumores e suas respectivas margens cirúrgicas devido a sua relação com SET, PP2A e hnRNP K. Os resultados mostraram acúmulo da c-Myc fosforilada e total nas amostras tumorais, o que coincidiu com aumento de SET e hnRNP K. Com relação à proteína MARK3, observou-se sua redução no tumor e menor sobrevida livre de doença. Foi realizado ensaio de RNA de interferência (RNAi) contra hnRNP K e SET em linhagem de carcinoma oral (HN13). A redução da proteína SET por RNAi levou a redução significativa da hnRNP K, enquanto a hnRNP K gerou menor efeito na proteína SET, sugerindo um efeito regulatório na expressão ou manutenção da hnRNP K pela SET na célula tumoral. A interferência contra a hnRNP K também reduziu a proliferação celular tumoral. Em conclusão, o aumento da proteína SET está associado à desmoplasia em HNSCC e pode ser um potencial marcador específico para essa condição. hnRNP K e MARK3 podem servir como potenciais marcadores em HNSCC e ajudar a identificar um subgrupo de pacientes com pobre prognóstico. A hnRNPK exerce efeito positivo na proliferação da célula tumoral. SET e hnRNP K podem atuar como fatores oncogênicos favorecendo o aumento de c-Myc. / The head and neck cancers constitute a major public health problem due to the high incidence and some types are associated with behavioral factors such as consumption of alcohol and tobacco. Despite these data, the disease, especially in its early stage can be cured and some types can be prevented. Therefore, there is a need to identify and validate new biomarkers in head and neck cancer, with applications in prognosis and selection of therapies most appropriate. Accordingly, the objectives of this study were validation of the profile of three proteins, SET, hnRNP K and MARK3 in tumors of head and neck, and verify the potential application as markers for diagnosis and prognosis in HNSCC, and suggest a role for these proteins in tumorigenesis. We analyzed 22 samples of head and neck tumors by western blotting (WB) and 96 samples (91 tumors, 4 biopsies and 1 control) arranged in duplicate in the tissue microarray slide, obtained in Brazil and assigned by the GENCAPO Group, by immunohistochemistry (IHC). The data were correlated with all clinical and pathological parameters and prognosis of patients with HNSCC for a period of 48 months. The results obtained by WB and IHC showed the SET accumulation and fragmentation and hnRNP K nuclear and cytoplasmic accumulation in tumor compared to the surgical margin and normal tissue. The hnRNPK prognostic value has been associated with overall survival of patients. The c-Myc protein and its phosphorylated form were analyzed in tumor and surgical margins samples due to its relationship with SET, PP2A and hnRNP K. The results showed accumulated total and phosphorylated c-Myc in tumor samples, which was coincided with increase in SET and hnRNP K. Regarding the protein MARK3 was observed its reduction in tumor and lower disease-free survival. RNA interference (RNAi) against hnRNP K and SET were performed in oral squamous cell carcinoma line (HN13). SET protein reduction by RNAi led to significant reduction of hnRNP K, and hnRNP K showed a minor effect on SET protein, suggesting a regulatory effect on expression or maintenance of hnRNP K by SET in tumor cells. Interference against hnRNP K also reduced tumor cell proliferation. In conclusion, increased SET protein is associated with desmoplasia in HNSCC and may be a potential specific marker for this condition. hnRNP K and MARK3 can serve as potential markers in HNSCC and help identify a subgroup of patients with poor prognosis. The hnRNPK must act a positive effect on cell proliferation of the tumor. SET and hnRNP K may act as oncogenic factors contributing for c-Myc activity.