Many current efforts in cancer research focus on understanding the molecular mechanisms driving oncogenesis and to advance molecular diagnostics and targeted therapeutics. The MYC oncoprotein is estimated to be deregulated in over 50% of human cancers, and its deregulation is often associated with aggressive disease and poor patient outcomes. While the ability of MYC to promote cellular transformation is well established, a better understanding of the mechanisms promoting MYC-mediated tumorigenesis is essential. While MYC has been shown to undergo a number of post-translational modifications (PTMs), our current understanding of biological significance of these modifications is largly limited to two phosphorylation sites located in the N-terminal domain of the protein. Our work, therefore, aimed to further our understanding of how PTMs regulate MYC-dependent transformation. To this end we have identified and characterized three novel human cell line models of MYC-dependent transformation: MCF10A, SH-EP Tet21/N-Myc, and LF1/TERT/LT/ST cells. Using a combination of these novel models and classic systems, we have evaluated point mutants of MYC at key serine/threonine and lysine residues for their ability to influence MYC-dependent transformation. Using a six lysine to arginine substitution mutant, we have identified and chacterized six C-terminal lysines to be important for the negative regulation of MYC activity. We have additionally demonstrated for the first time that MYC can undergo SUMOylation at one of the lysines in this region. We further completed a functional and transcriptional characterization of MYC phosphorylation mutants. We have assigned biological significance to previously identified phosphorylation sites through the characterization of two mutants that have increased transformation potential over wild-type MYC. Expression array analysis identified gene expression changes both common to deregulated MYC and unique to the different gain-of-function phosphorylation mutants. Combined, this work has advanced our understanding of several of the mechanisms that may regulate MYC-induced transformation.

Myc is an oncogene that is commonly deregulated in human cancers. Mechanistic studies reveal that Myc is a transcription factor that interacts with a protein partner called Max. Heterodimerization and the formation of the Myc:Max complex enables Myc:Max to bind to the E-box and subsequently regulate the activation and repression of Myc target genes. Since regulation of its target genes are essential for Myc to drive transformation, the Myc and Max interaction has been targeted in mouse model studies to determine whether the oncogenic activity of Myc can be inhibited. Surprisingly, these studies reveal that targeting Myc is not only possible but a powerful way to suppress tumour growth. Since a better understanding of how Myc carries out its biological functions makes the possibility of targeting Myc a reality, it is essential to investigate and study the mechanisms of how Myc promotes tumourigenesis. In the first part of this thesis, we investigate the idea that the N-terminal end of Myc may be post-translationally modified and this modification may dictate Myc activities. Indeed we report here that the N-terminal end of Myc can be ubiquitylated as well as acetylated, and that the loss of these modifications results in a decrease in Myc activities. Furthermore, we characterize Mdm2 as a potential E3 ubiquitin ligase that may ubiquitylate the N-terminal end of Myc. In the second part of this thesis, we investigate CUL7 as a novel oncogene that may inhibit Myc-potentiated apoptosis and cooperate with Myc in transformation. Indeed, CUL7 is a novel p53 interacting protein that inhibits Myc potentiated apoptosis through the inhibition of p53. We have also characterized CUL7 to be overexpressed in primary human lung cancers, and a higher level of CUL7 expression associates with short-term survival of lung cancer patients. Through the better understanding of the enzymes that post-translationally modify the N-terminal end of Myc and proteins, such as CUL7, that can cooperate with Myc to drive tumourigenesis, we may begin to devise ways to target and control deregulated Myc in cancer cells.

Appendix on leaves 193-197. / Additional notes pasted on back fly-leaf. / Copies of authors previously published articles in pocket inside back cover. / Bibliography: leaves 147-192. / 197, [114] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Enforced-expression of various forms of Myb and Kit in primary murine haemopoietic cells were investigated to determine the functions of these two oncoproteins separately and in synergy in vitro. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1998?

Univ., FB Chemie, Diss., 2001--Marburg

The transcription factor Myc interacts with several co-factors to regulate growth and proliferationand thereby enables normal animal development. Deregulation of Myc is associated witha wide range of human tumors. Myc binds to DNA together with its dimerization partner Max, preferentially to canonical E-box motifs, but this sequence-specific interaction is probably not sufficient for Myc’s binding to target genes. In this work, the PAF1 complex was characterized as a novel co-factor of Myc in Drosophila melanogaster. All components of the complex are required for Myc’s recruitment to chromatin, but the subunit Atu has the strongest effect on Myc's binding to target genes through ist direct physical interaction with Myc. Unexpectedly, the impact of Atu depletion on the Expression of Myc target genes was weak compared to its effect on Myc binding. However, the influence of Atu becomes more prominent in situations of elevated Myc levels in vivo . Mycrepressed as well as Myc-activated targets are affected, consistent with the notion that Myc recruitment is impaired. An independent set of analyses revealed that Myc retains substantial activity even in the complete absence of Max. The overexpression of Myc in Max0 mutants specifically blocks their pupariation without affecting their survival, which raised the possibility that Myc might affect ecdysone biosynthesis. This connection was studied in the second part of this Thesis which showed that Myc inhibits the expression of ecdysteroidogenic genes and thereby the production of ecdysone. Myc most likely affects the signaling pathways (PTTH and insulin signaling) upstream of the PG, the organ where ecdysone is produced. By combining existing ChIPseq, RNAseq and electronic annotation data, we identified five potential Maxindependent Myc targets and provided experimental data that they might be involved in Myc's effect on Max mutant animals. Together our data confirm that some Myc functions are Max-independent and they raise the possibility that this effect might play a role during replication. / Der Transkriptionsfaktor Myc interagiert mit verschiedenen Cofaktoren, um Wachstum und Proliferation zu regulieren, was die normale Entwicklung von Tieren ermöglicht. Die Fehlreguliereung von Myc wird mit einer großen Anzahl menschlicher Tumore in Verbindung gebracht. Myc bindet gemeinsam mit seinem Dimerisationspartner Max an DNA, bevorzugt an kanonische E-Box Motive. Allerdings ist diese sequenz-spezifische Interaktion wahrscheinlich nicht ausreichend für die Bindung von Myc an Zielgene. In dieser Arbeit wurde der PAF1 Komplex als ein neuartiger Cofaktor von Myc in Drosophila melanogaster charakterisiert. Alle Komponenten des Komplexes sind für die Rekrutierung von Myc an Chromatin notwendig, jedoch hat die Untereinheit Atu, durch ihre direkte physische Interaktion mit Myc, den stärksten Effekt auf die Bindung von Myc an Zielgene. Verglichen mit dem Effekt auf die Bindung von Myc hatte die Depletion von Atu nur einen schwachen Einfluss auf die Expression der Myc Zielgene. In vivo ist der Einfluss von Atu stärker ausgeprägt in Situationen in denen die Myc Proteinlevel erhöht sind. Sowohl Myc-reprimierte als auch Myc-aktivierte Gene sind dadurch betroffen. Dies stimmt mit der Entdeckung überein, dass die Rekrutierung von Myc beeinträchtigt ist. Unabhängige Versuche haben gezeigt, dass Myc eine deutliche Aktivität behält auch bei vollständiger Abwesenheit von Max. Die Überexpression von Myc in Max0 Mutanten verhindert deren Verpuppung ohne ihr Überleben zu beeinträchtigen. Dies führt zu der Vermutung, dass Myc einen Einfluss auf die Biosynthese von Ecdyson hat. Diese Verbindung wurde im zweiten Teil der Arbeit untersucht und hat gezeigt, dass Myc die Expression von Genen, die an der Ecdyson-Synthese beteiligt sind, verhindert und dadurch die Produktion von Ecdyson selbst. Myc wirkt bevorzugt auf die Signalwege (PTTH und Insulin Signalkaskade) oberhalb der Prothorakaldrüse, dem Organ in dem Ecdyson produziert wird. Durch die Kombination von ChIPseq, RNAseq und der Auswertung elektronischer Daten wurden von uns fünf potentielle Max-unabhängige Zielgene von Myc identifiziert. Des weiteren haben experimentelle Daten gezeigt, dass diese in Zusammenhang mit dem Effekt von Myc auf Max0 Mutanten stehen. Zusammenfassend haben unsere Daten bestätigt, dass einige Funktionen von Myc Max-unabhängig sind und es besteht die Möglichkeit, dass dieser Effekt eine Rolle während der Replikation spielen könnte.

Der niedermolekulare Inhibitor JQ1 bindet an der Bromodomäne von BRD4, ein auf epigenetischer Ebene agierendes Protein. Der antiproliferative Effekt von JQ1 wurde bisher bei verschiedenen Tumorentitäten vor allem des lymphatischen und blutbildenden Systems gezeigt. In dieser Arbeit wurde der antiproliferative Effekt von JQ1 an fünf humanen kolorektalen Karzinomzelllinien im Vergleich zu nicht transformierten Kontrollzellen (Fibroblasten) in Normoxie, Hypoxie und in der Langzeitkultur nachgewiesen. Außerdem verringerte JQ1 die Expression von MYC auf Protein- und mRNA-Ebene und steigerte die Transkription des durch MYC negativ regulierten Zielegens p21. Diese Steigerung korrelierte mit einem Zellzyklusarrest in der G0/G1-Phase in vier von fünf kolorektalen Karzinomzelllinien. / The small molecule inhibitor JQ1 binds to the bromodomain of BRD4, a protein acting on the epigenetic level. The antiproliferative effect of JQ1 has been shown in various tumor entities, especially in lymphatic and hematopoietic systems. In this study, the antiproliferative effect of JQ1 was demonstrated in five human colorectal carcinoma cell lines compared to non-transformed control cells (fibroblasts) in normoxia, hypoxia and long-term culture. In addition, JQ1 reduced expression of MYC at the protein and mRNA levels and increased transcription of MYC-negatively regulated target p21. This increase correlated with a cell cycle arrest in the G0/G1 phase in four of five colorectal carcinoma cell lines.

La leucémie aiguë lymphoblastique (LAL-T) est une hémopathie maligne qui représente 10 à 15% des LAL pédiatriques et 25% des LAL de l’adulte. Bien que la prise en charge et le pronostic (rémission dans 80-85% des cas) des LAL se soient améliorés au cours des 10 dernières années en partie dû à une meilleure stratification thérapeutique de ces entités malignes, le tableau clinique et le devenir des patients atteints de LAL-T restent péjoratif avec environ 30% de rechute dans les 2 années qui suivent le diagnostic. Au cours de ces dernières années, des sous-types spécifiques de LAL-T associés à une valeur pronostique ont été décrits et des thérapies ciblées devraient pouvoir être proposées à l’avenir. Dans ce contexte, mon travail de thèse a permis de définir et de mieux comprendre les différents niveaux de dérégulation de MYC dans les LAL-T à travers l’analyse moléculaire et biochimique de MYC et de ses principaux régulateurs sur une large cohorte protocolaire de LAL-T pédiatriques et adultes. Tout d’abord, nous montrons que l'expression de MYC est très variable et que des niveaux d'expression élevés sont observés dans de nombreux cas en absence de mutations NOTCH1/FBXW7. De plus, nos travaux mettent en évidence que la dérégulation post-traductionnelle de MYC, via l'axe PI3K/AKT à travers l'inactivation de PTEN, constitue une voie majeure d'activation de MYC dans les LAL-T. Ainsi, l'ensemble de ces résultats confirment la pertinence d’envisager des stratégies thérapeutiques ciblant MYC pour le traitement des LAL-T. Mon projet de thèse a également consisté en la génération d’un modèle murin original permettant de suivre les clones tumoraux surexprimant Myc depuis les étapes de développement (pré-)tumoral les plus précoces jusqu’aux étapes finales de progression maligne. / T-cell Acute Lymphoblastic Leukemia (T-ALL) are malignant proliferations of thymocytes, which represent 10-15% of pediatric and 25% of adult ALL. Despite indisputable therapeutic progress, T-ALLs remain of poor prognosis. Patients often present with a high tumor load accompanied by a rapid disease progression, and about 30% of cases relapse within the first 2 years following diagnosis. It is now clear that significant improvements in therapy will require a more accurate knowledge of the oncogenes involved, as well as their oncogenic role within complex functional networks. In this context, my PhD project was focused on the understanding of the regulation of MYC in T-ALL. We demonstrate that MYC expression is highly variable and that high MYC expression levels can be generated independently of NOTCH1 pathway. Furthermore, we show that posttranscriptional deregulation of MYC constitutes a major alternative pathway of MYC activation in T-ALL, operating partly via the PI3K/AKT axis through down regulation of PTEN. Altogether, our results lend further support to the significance of therapeutic targeting of MYC in T-ALL pathogenesis. The second part of my project was to generate an original transgenic mouse model designed to “track” inducible MYC+ clones from the earliest steps of (pre-)malignant development to the onset of leukemia.

No description available.

Das Proto-Onkoprotein Myc ist an der Entstehung und Aufrechterhaltung einer Vielzahl humaner Tumore entscheidend beteiligt. In der vorliegenden Arbeit wurde Serin 227 in Fbw7 als Ziel für eine PI3K-abhängige Phosphorylierung identifiziert. Diese Phosphorylierung führt zur Stabilisierung von Fbw7 und steigert die Fähigkeit von Fbw7, Substratproteine zu ubiquitinieren und abzubauen. Um die Bedeutung von Usp28 in der Myc-induzierten Tumorentstehung und in der normalen Gewebehomöostase zu untersuchen, wurde ein konditionales Knockout-Mausmodell für Usp28 charakterisiert. Mäuse mit einer Keimbahndeletion von Usp28 sind lebensfähig, fertil und phänotypisch unauffällig. Weder in Organen der Usp28-negativen Tiere, noch in entsprechenden murinen embryonalen Fibroblasten kann eine Destabilisierung von Myc festgestellt werden. Allerdings zeigen Fibroblasten mit heterozygotem Usp28-Verlust einen Proliferationsdefekt und in Eμ-Myc-Lymphomen dieses Genotyps werden tendenziell niedrigere Myc-Proteinmengen gefunden. Das tumorfreie Überleben ist bei den Eμ-Myc; Usp28 +/- Tieren verlängert. / The proto-oncoprotein Myc is involved in the genesis and maintenance of a large fraction of human tumors. In this work, I identified serine 227 in Fbw7 as a target for PI3K-dependent phosphorylation. The phosphorylation leads to stabilization of Fbw7 and enhances its ability to promote ubiquitination and degradation of its substrates. To investigate the role of Usp28 in Myc-dependent tumorigenesis and in tissue homeostasis I characterized a Usp28 conditional knockout mouse model. Mice with a germline deletion of Usp28 are viable, fertile and phenotypically normal. No decrease in Myc protein levels could be detected in organs or embryonic fibroblasts of Usp28- knockout mice. Surprisingly, embryonic fibroblasts with a heterozygous Usp28 deletion showed a proliferative defect and Eμ-Myc lymphomas of this genotype showed a tendency to reduced Myc protein levels, corresponding to a longer tumorfree survival of these animals.

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