Murine plasmacytoma is induced by plastic implants, injection of paraffin oil or pristane, or through viral infection, and Myc is invariably overexpressed in the tumour cells. Although translocation and juxtaposition of the Myc locus to an immunoglobulin locus is the prominent nonrandom cytogenetic aberration observed, the significance of other karyotypic instabilities in murine plasmacytoma is not clear, including the previously observed occurrence of trisomy 11. As well as identifying new cytogenetic mutations in murine plasmacytomagenesis, this study provides evidence for their combined and sequential accumulation that may offer new parallels to human B-cell malignancies. Plasmacytomas were induced in Balb/c Rb6.15 mice by intraperitoneal (i.p.) pristane injection prior to infection with the ABL-MYC retrovirus, and confirmed by histological examination. Spectral karyotype analysis of tumour samples identified frequent aneuploidy, tetraploidy, and amplification of chromosomes 11, 12 and 16. In contrast, control mice treated by i.p. pristane injection did not develop plasmacytoma, and lipopolysaccharide-stimulated splenocytes from control mice had mainly normal diploid karyotypes. However, karyotypic instability in a minority of splenocytes indicated that control mice showing no signs of plasmacytoma development nevertheless are prone to numerical and structural cytogenetic mutations that may possibly result in plasmacytoma initiation and progression under favourable conditions, such as infection with ABL-MYC virus with the resulting high expression of v-abl and Myc in target cells. These results indicate the possible existence of proto-oncogenes present on murine chromosomes 11, 12, and 16 that are important for plasmacytoma initiation and/or progression. There are also indications that T(1;6) and monosomy of the X chromosome may also play roles in plasmacytomagenesis, and that trisomy 12 may only occur in cells with pre-existing nonrandom mutations, thereby acting as a late mutation event. As other experimental models of murine plasmacytoma have not shown a similar karyotypic etiology, there appears to be several possible redundant cytogenetic mutation events that lead to plasmacytoma. Also, as tumours in this study present various combinations of the aforementioned amplified chromosomes, their combined amplification may serve redundant purposes as well. / May 2008

Reproduction de : Thèse doctorat : Pharmacie. Biologie moléculaire et physiologie : Reims : 2005. / Bibliogr. p.180-217. Index.

Murine plasmacytoma is induced by plastic implants, injection of paraffin oil or pristane, or through viral infection, and Myc is invariably overexpressed in the tumour cells. Although translocation and juxtaposition of the Myc locus to an immunoglobulin locus is the prominent nonrandom cytogenetic aberration observed, the significance of other karyotypic instabilities in murine plasmacytoma is not clear, including the previously observed occurrence of trisomy 11. As well as identifying new cytogenetic mutations in murine plasmacytomagenesis, this study provides evidence for their combined and sequential accumulation that may offer new parallels to human B-cell malignancies. Plasmacytomas were induced in Balb/c Rb6.15 mice by intraperitoneal (i.p.) pristane injection prior to infection with the ABL-MYC retrovirus, and confirmed by histological examination. Spectral karyotype analysis of tumour samples identified frequent aneuploidy, tetraploidy, and amplification of chromosomes 11, 12 and 16. In contrast, control mice treated by i.p. pristane injection did not develop plasmacytoma, and lipopolysaccharide-stimulated splenocytes from control mice had mainly normal diploid karyotypes. However, karyotypic instability in a minority of splenocytes indicated that control mice showing no signs of plasmacytoma development nevertheless are prone to numerical and structural cytogenetic mutations that may possibly result in plasmacytoma initiation and progression under favourable conditions, such as infection with ABL-MYC virus with the resulting high expression of v-abl and Myc in target cells. These results indicate the possible existence of proto-oncogenes present on murine chromosomes 11, 12, and 16 that are important for plasmacytoma initiation and/or progression. There are also indications that T(1;6) and monosomy of the X chromosome may also play roles in plasmacytomagenesis, and that trisomy 12 may only occur in cells with pre-existing nonrandom mutations, thereby acting as a late mutation event. As other experimental models of murine plasmacytoma have not shown a similar karyotypic etiology, there appears to be several possible redundant cytogenetic mutation events that lead to plasmacytoma. Also, as tumours in this study present various combinations of the aforementioned amplified chromosomes, their combined amplification may serve redundant purposes as well.

Murine plasmacytoma is induced by plastic implants, injection of paraffin oil or pristane, or through viral infection, and Myc is invariably overexpressed in the tumour cells. Although translocation and juxtaposition of the Myc locus to an immunoglobulin locus is the prominent nonrandom cytogenetic aberration observed, the significance of other karyotypic instabilities in murine plasmacytoma is not clear, including the previously observed occurrence of trisomy 11. As well as identifying new cytogenetic mutations in murine plasmacytomagenesis, this study provides evidence for their combined and sequential accumulation that may offer new parallels to human B-cell malignancies. Plasmacytomas were induced in Balb/c Rb6.15 mice by intraperitoneal (i.p.) pristane injection prior to infection with the ABL-MYC retrovirus, and confirmed by histological examination. Spectral karyotype analysis of tumour samples identified frequent aneuploidy, tetraploidy, and amplification of chromosomes 11, 12 and 16. In contrast, control mice treated by i.p. pristane injection did not develop plasmacytoma, and lipopolysaccharide-stimulated splenocytes from control mice had mainly normal diploid karyotypes. However, karyotypic instability in a minority of splenocytes indicated that control mice showing no signs of plasmacytoma development nevertheless are prone to numerical and structural cytogenetic mutations that may possibly result in plasmacytoma initiation and progression under favourable conditions, such as infection with ABL-MYC virus with the resulting high expression of v-abl and Myc in target cells. These results indicate the possible existence of proto-oncogenes present on murine chromosomes 11, 12, and 16 that are important for plasmacytoma initiation and/or progression. There are also indications that T(1;6) and monosomy of the X chromosome may also play roles in plasmacytomagenesis, and that trisomy 12 may only occur in cells with pre-existing nonrandom mutations, thereby acting as a late mutation event. As other experimental models of murine plasmacytoma have not shown a similar karyotypic etiology, there appears to be several possible redundant cytogenetic mutation events that lead to plasmacytoma. Also, as tumours in this study present various combinations of the aforementioned amplified chromosomes, their combined amplification may serve redundant purposes as well.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 122-177).

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 122-177). Also available by subscription via World Wide Web.

Im Neuroblastom ist die Amplifikation des MYCN-Gens, eines Mitglieds der MYC-Onkogenfamilie, mit einer ungünstigen Prognose assoziiert. Der von dem Gen kodierte Transkriptionsfaktor N-Myc ist für die Proliferation der MYCN-amplifizierten Neuroblastomzelllinien notwendig und seine Depletion oder Destabilisierung führen zum Proliferationsarrest (Otto et al., 2009). Da N-Myc auf Proteinebene durch die Interaktion mit der mitotischen Kinase Aurora-A stabilisiert wird, bewirkt deren Depletion oder die Hemmung der Interaktion der beiden Proteine mittels spezieller Aurora- A-Inhibitoren (z.B. MLN8054 und MLN8237) ebenso eine Hemmung der Proliferation – in vitro und in vivo (Brockmann et al., 2013). Bisher ist jedoch unklar, über welchen Mechanismus Aurora-A die Stabilisierung von N-Myc erreicht, die Kinaseaktivität spielt hierbei jedoch keine Rolle (Otto et al., 2009). Eine Möglichkeit stellt die Rekrutierung von Usps dar, die das angehängte Ubiquitinsignal so modifizieren, dass die Erkennung und der Abbau des Proteins durch das Proteasom verringert werden. In der vorliegenden Arbeit wurde die Wirkung von Usp7 und Usp11 auf die Stabilität von N-Myc untersucht. Für beide konnte in Immunpräzipitationen die Interaktion mit N-Myc gezeigt werden. Ebenso erhöhten beide Proteasen in Überexpressionsexperimenten die vorhandene Menge an NMyc. Die Depletion von Usp7 mittels shRNAs führte in IMR-32 zu einem Arrest in der G1-Phase und zur Differenzierung der Zellen. Gleichzeitig wurden stark erniedrigte mRNA- und Proteinmengen von N-Myc und Aurora-A nachgewiesen. Es konnte jedoch nicht eindeutig gezeigt werden, ob die beobachteten zellulären Effekte durch eine vermehrte proteasomale Degradation von N-Myc begründet sind oder ob dabei die veränderte Regulation weiterer Zielproteine von Usp7 eine Rolle spielt. Die Depletion von Usp11 mit shRNAs bewirkte eine Abnahme der N-Myc-Mengen auf posttranslationaler Ebene. Somit stellen beide Usps vielversprechende Angriffspunkte einer gezielten Therapie in MYCN-amplifizierten Neuroblastomen dar und sollten deshalb Gegenstand weiterführender Untersuchungen sein. Über welche Proteindomäne in N-Myc die Interaktion mit Aurora-A stattfindet ist nicht bekannt. Eine mögliche Pseudosubstratbindungssequenz in Myc-Box I (Idee Richard Bayliss, University of Leicester) wurde in der vorliegenden Arbeit untersucht. Durch Mutation dieser Sequenz sollte die Bindung von Aurora-A unmöglich gemacht werden. Allerdings wurde die erwartete Abnahme der Stärke der Interaktion von Aurora-A und N-Myc durch die Mutation ebensowenig beobachtet wie eine verringerte Stabilität. Die Regulation der Phosphorylierung von N-Myc im Verlauf des Zellzyklus wurde durch die Mutation beeinträchtigt. Wie diese Veränderung exakt zu begründen ist bedarf weiterer Experimente / Neuroblastomas with an amplification of the MYCN-gene, a member of the MYC-oncogene family, are associated with a poor prognosis. The transcription factor encoded by this gene, N-Myc, is essential for the proliferation of MYCN-amplified neuroblastoma cell lines and its depletion or destabilization leads to an arrest of proliferation (Otto et al., 2009). Since N-Myc is stabilized by the interaction with the mitotic kinase Aurora-A, the depletion of the kinase or the inhibition of the interaction with N-Myc using a special class of Aurora-A inhibitors (e.g. MLN8054 and MLN8237) inhibits proliferation – in vitro and in vivo (Brockmann et al., 2013). Up to date it is not known by which mechanism Aurora-A is able to stabilize N-Myc preventing it from Fbxw7-mediated proteasomal degradation, interestingly the Aurora-A kinase activity is not necessary (Otto et al., 2009). One possible explanation is the recruitment of Usps, which modify the attached ubiquitin signal and therefore reduce the recognition and degradation of the protein by the proteasome. In this thesis the influence of Usp7 and Usp11 on N-Myc stability was studied. For both the interaction with N-Myc was shown in immune precipitations. Furthermore the overexpression of both proteases increased the amount of N-Myc protein in transfection experiments. The depletion of Usp7 via shRNAs caused the arrest of IMR-32 cells in G1-phase and the differentiation of the cells. Simultaneously strongly reduced amounts of N-Myc and Aurora-A mRNA and proteins were observed. However it could not be shown that the observed effects were mediated by an increased proteasomal degradation of N-Myc and not via the changed regulation of other targets of Usp7. The depletion of Usp11 led to a decrease of the N-Myc amounts, whereas mRNA-levels were unaffected. Thus both Usps are promising targets for a targeted therapy of MYCN-amplified neuroblastomas and the underlying mechanism should be the object of further research. Furthermore the N-Myc domain binding to Aurora-A still remains to be idientified. A possible pseudosubstrate binding site in Myc-Box I (idea of Richard Bayliss, University of Leicester) was investigated in this thesis. To inhibit the possible binding of Aurora-A to this site, two lysines in Myc-Box I were mutated to glutamate (KK51EE). Nonetheless neither the expected decrease of the intensity of interaction of N-Myc and Aurora-A was observed nor was a decrease of the stability of N-Myc. The regulation of the phosphorylation of N-Myc during the cell cycle was changed through the mutagenesis however. It must be clarified in further experiments, what the reasons for this change are.

Interactions between DNA and ligands are important in the rational design of drugs and in research into DNA function. In particular, the interaction of DMZ with DNA structures named “G-quadruplexes” was considered. G-quadruplexes are structures present in telomeres and several oncogenes. The main purpose of this project was to provide a computational tool to study DNA ligand interactions using a variety of molecular modeling techniques that include molecular docking, molecular dynamics simulations (MD) and MM/PBSA (Molecular Mechanics/Poisson Boltzmann Surface Area). We investigated the binding modes and binding affinities of DMZ with c-MYC G-quadruplexes (G4s). We found that the conformation and structural design of the quadruplex can dramatically influence the binding profiles of the ligand. The binding free energies for each site were estimated by the MM/PBSA method. The binding of small molecules to DNA can result in the disruption of oncogene transcription, making it an effective anticancer strategy.

The expression of the MYC proto-oncogene is elevated in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC). Previous findings in PDAC have shown that this increased MYC expression mediates immune evasion and promotes S-phase progression. How these functions are mediated and whether a downstream factor of MYC mediates these functions has remained elusive. Recent studies identifying the MYC interactome revealed a complex network of interaction partners, highlighting the need to identify the oncogenic pathway of MYC in an unbiased manner. In this work, we have shown that MYC ensures genomic stability during S-phase and prevents transcription-replication conflicts. Depletion of MYC and inhibition of ATR kinase showed a synergistic effect to induce DNA damage. A targeted siRNA screen targeting downstream factors of MYC revealed that PAF1c is required for DNA repair and S-phase progression. Recruitment of PAF1c to RNAPII was shown to be MYC dependent. PAF1c was shown to be largely dispensable for cell proliferation and regulation of MYC target genes. Depletion of CTR9, a subunit of PAF1c, caused strong tumor regression in a pancreatic ductal adenocarcinoma model, with long-term survival in a subset of mice. This effect was not due to induction of DNA damage, but to restoration of tumor immune surveillance. Depletion of PAF1c resulted in the release of RNAPII with transcription elongation factors, including SPT6, from the bodies of long genes, promoting full-length transcription of short genes. This resulted in the downregulation of long DNA repair genes and the concomitant upregulation of short genes, including MHC class I genes. These data demonstrate that a balance between long and short gene transcription is essential for tumor progression and that interference with PAF1c levels shifts this balance toward a tumor-suppressive transcriptional program. It also directly links MYC-mediated S-phase progression to immune evasion. Unlike MYC, PAF1c has a stable, known folded structure; therefore, the development of a small molecule targeting PAF1c may disrupt the immune evasive function of MYC while sparing its physiological functions in cellular growth. / Die Expression des MYC-Proto-Onkogens ist bei einem großen Teil der Patienten mit duktalem Adenokarzinom der Bauchspeicheldrüse (PDAC) erhöht. Bisherige Erkenntnisse in der Erforschung des ankreaskarzinoms zeigen, dass die erhöhte MYCExpression die Umgehung des Immunsystems bewirkt und die Progression der S-Phase fördert. Wie diese Funktionen vermittelt werden und ob ein nachgeschalteter Faktor von MYC für diese Funktion verantwortlich ist, blieb jedoch bisher ungeklärt. Jüngste Studien zur Identifizierung des MYC-Interaktoms haben ein sehr komplexes Netzwerk an Interaktionspartnern von MYC aufgedeckt, was die Notwendigkeit unterstreicht, die onkogenen Eigenschaften von MYC und seinen Interaktionspartnern unvoreingenommen und genau zu untersuchen. In dieser Arbeit konnte gezeigt werden, dass MYC die genomische Stabilität während der S-Phase herstellt und Konflikte zwischen Transkription und Replikation verhindert. Die Depletion von MYC und die Hemmung der ATR-Kinase zeigten bei der Induktion von DNA Schäden eine synergistische Wirkung. Ein siRNA-Screen, der Gene beinhaltete, die MYC nachgeschaltet sind, ergab, dass PAF1c für die DNA-Reparatur und die S-PhasenProgression erforderlich ist. Es zeigte sich außerdem, dass die Rekrutierung von PAF1c an RNAPII von MYC abhängig ist. Für die Zellproliferation und die Regulierung von MYCZielgenen ist PAF1c jedoch weitgehend entbehrlich. Es konnte gezeigt werden, dass die Depletion von CTR9, einer Untereinheit von PAF1c, in einem murinen Modell des duktalen Adenokarzinoms der Bauchspeicheldrüse zu einer starken Tumorregression mit langfristigem Überleben einiger Mäuse führte. Diese Wirkung war nicht auf die Induktion von DNA-Schäden zurückzuführen, sondern auf die Wiederherstellung der Immunüberwachung des Tumors. Die Deletion von PAF1c führte zu einer Umverteilung von RNAPII und Trankriptionselongationsfaktoren wie SPT6, von langen Genen hin zu kurzen Genen. Dadurch wurden lange Gene wie zum Beispiel DNA Reparaturgene nicht vollständig transkribiert, kurze Gene wie MHC-Klasse-I-Gene hingegen schon. Diese Daten zeigen, dass ein Gleichgewicht zwischen der Transkription langer und kurzer Gene für die Tumorprogression wichtig ist und dass eine Verminderung der PAF1c-Konzentration dieses Gleichgewicht in Richtung eines tumorsuppressiven Transkriptionsprogramms verschiebt. Außerdem besteht ein direkter Zusammenhang zwischen der MYCvermittelten S-Phasen-Progression und der Umgehung des Immunsystems. Im Gegensatz zu MYC verfügt PAF1c über eine stabile und gut bekannte gefaltete Struktur. Daher könnte die Entwicklung eines kleinen Moleküls, das PAF1c hemmt, die Funktion von MYC zur Umgehung des Immunsystems stören und gleichzeitig seine physiologischen Funktionen für das Zellwachstum nicht beeinträchtigen.

Deregulated expression of MYC oncoproteins is a driving event in many human cancers. Therefore, understanding and targeting MYC protein-driven mechanisms in tumor biology remain a major challenge. Oncogenic transcription in MYCN-amplified neuroblastoma leads to the formation of the MYCN-BRCA1-USP11 complex that terminates transcription by evicting stalling RNAPII from chromatin. This reduces cellular stress and allows reinitiation of new rounds of transcription. Basically, tumors with amplified MYC genes have a high demand on well orchestration of transcriptional processes-dependent and independent from MYC proteins functions in gene regulation. To date, the cooperation between promoter-proximal termination and transcriptional elongation in cancer cells remains still incomplete in its understanding. In this study the putative role of the dubiquitinase Ubiquitin Specific Protease 11 (USP11) in transcription regulation was further investigated. First, several USP11 interaction partners involved in transcriptional regulation in neuroblastoma cancer cells were identified. In particular, the transcription elongation factor A like 1 (TCEAL1) protein, which assists USP11 to engage protein-protein interactions in a MYCN-dependent manner, was characterized. The data clearly show that TCEAL1 acts as a pro-transcriptional factor for RNA polymerase II (RNAPII)-medi- ated transcription. In detail, TCEAL1 controls the transcription factor S-II (TFIIS), a factor that assists RNAPII to escape from paused sites. The findings claim that TCEAL1 outcompetes the transcription elongation factor TFIIS in a non-catalytic manner on chromatin of highly expressed genes. This is reasoned by the need regulating TFIIS function in transcription. TCEAL1 equili- brates excessive backtracking and premature termination of transcription caused by TFIIS. Collectively, the work shed light on the stoichiometric control of TFIIS demand in transcriptional regulation via the USP11-TCEAL1-USP7 complex. This complex protects RNAPII from TFIIS-mediated termination helping to regulate productive transcription of highly active genes in neuroblastoma. / Die deregulierte Expression von MYC Onkoproteinen ist ein zentrales Event in vielen huma-nen Krebsarten. Aus diesem Grund sind das Verständnis und die gezielte Bekämpfung MYC-getriebener Mechanismen in der Tumorbiologie nach wie vor eine große Herausforderung. In MYCN-amplifizierten Neuroblastomen führt eine übermäßig hohe Transkriptionsrate zur stress-bedingten Rekrutierung des MYCN-BRCA1-USP11-Komplexes. Dieser Komplex be-endet vorzeitig die Transkription, indem er RNAPII Moleküle vom Chromatin wirft. Durch diesen Mechanismus wird zellulärer Stress reduziert und ermöglicht dadurch einen erneuten Start der Transkription. Grundsätzlich stellen Tumoren mit einer Amplifikation von einem der MYC Proteine hohe Anforderungen an eine feine Abstimmung der einzelnen Schritte in der Transkription. Dies ist sowohl abhängig als auch unabhängig von den bereits beschriebe-nen Funktionen der MYC-Proteine in der Genregulation. Bis heute ist das Zusammenspiel zwischen promoter-proximaler Termination und transkriptioneller Elongation noch nicht vollständig aufgeklärt. In dieser Studie wurde eine potenzielle Rolle von USP11 in der Regulation der Transkription weitergehend untersucht. Zunächst wurden mehrere Interaktionspartner von USP11, die an der Regulation der Transkription in Neuroblastom Krebszellen beteiligt sind, identifiziert. Es wurde insbesondere das Transcription Elongation Factor A Like 1 (TCEAL1) Protein charak-terisiert. Dieses Protein unterstützt USP11 dabei, Protein-Protein-Interaktionen MYCN-vermittelt einzugehen. Die Daten zeigen, dass TCEAL1 als pro-transkriptioneller Faktor für die RNA-Polymerase II (RNAPII) -vermittelte Transkription fungiert. Genauer, TCEAL1 kontrolliert den Transkriptionsfaktor S-II (TFIIS), einen Faktor, der der RNAPII dabei hilft, die Transkription nach einem kurzen Pausieren („pausing“) fortzusetzen. Die Ergebnisse zei-gen, dass TCEAL1 den Elongationsfaktor TFIIS auf nicht-katalytische Weise von dem Chromatin von hochexprimierten Genen verdrängt. Dies ist darin begründet, dass die Funkti-on von TFIIS bei der Transkription reguliert werden muss. TCEAL1 gleicht übermäßiges Zurückwandern der RNAPII und die vorzeitige Beendigung der Transkription, das durch TFIIS vermittelt wird, aus. Diese Arbeit gibt Aufschluss über die stöchiometrische Kontrolle des TFIIS-Bedarfs bei der Transkriptionsregulation durch den USP11-TCEAL1-USP7-Komplex. Dieser Komplex schützt die RNAPII vor der TFIIS-vermittelter Termination der Transkription und trägt zur Regulierung einer produktiven Transkription hochaktiver Gene im Neuroblastom bei.