The COP9 signalosome (CSN) contains eight-subunits and is a highly conserved protein complex implicated in ubiquitin-mediated proteolysis. Jun activation domain-binding protein 1 (Jab1) is the fifth component of CSN (CSN5) with a molecular weight of 38 kDa. Jab1 overexpression is observed in many human cancers, but there is no clear evidence how Jab1 contributes to malignant transformation in human cancers. Bcr-Abl is a cytoplasmic chimeric oncoprotein produced by Philadelphia chromosome translocation and found in more than 90% of patients with chronic myelogenous leukemia (CML). Recent studies have shown that the Jab1/COP9 signalosome subcomplex is a downstream mediator of Bcr-Abl kinase and may facilitate cell-cycle progression. In this study, we found that inhibition of Bcr-Abl kinase by STI-571 downregulated 50% of full length human Jab1 promoter activity and gene expression. Promoter deletion assay indicated that the responsive element for Bcr-Abl is located between -405/-223 region of human Jab1 promoter. Treatment of LY294002 also reduced Jab-405/-223 promoter activity and Jab1 expression. Promoter mutagenesis assay and ChIP assay suggest that Bcr-Abl stimulated both £]-catenin /TCF complex and STAT1 bind to the consensus binding sites of Jab-405/-223 promoter. Taken together, Bcr-Abl oncogene may regulate Jab1 via PI3K/AKT signal pathway, £]-catenin /TCF and STAT1 transcription factors.

Die vorliegende Arbeit handelt von einer in vitro Untersuchung der Stoffwechselveränderun-gen durch die Expression von BCR/ABL bei BaF3-Zellen, einer murinen, IL-3-abhängigen B-Zelllinie. Die Zellen wurden in nährstoffreichem Standard- und in durch Titrationen ermittel-tem nährstoffarmem Minimalmedium auf unterschiedliche Stoffwechselaktivität in Abhän-gigkeit von BCR/ABL-Expression untersucht sowie auf die zusätzliche Beeinflussbarkeit durch IL-3. Danach wurden vergleichend zwischen den 2 Zelllinien (BaF3 und BaF3-BCR/ABL) im Minimalmedium und im Standardmedium Metabolite wie Glukose, Laktat und Aminosäuren bestimmt, wobei BaF3-BCR/ABL sowohl mit als auch ohne IL-3 kultiviert wur-de. Um den Einfluss von Nährstoffrestriktion auf die Therapie zu zeigen, wurden anschlie-ßend vergleichend in den beiden Medien die Tyrosinkinaseinhibitoren Imatinib und Nilotinib titriert. Die Ergebnisse zeigen, dass BaF3-BCR/ABL einen Wachstumsvorteil im Minimalmedium hat, welcher im Standardmedium nicht vorliegt. Während die bereits bekannte Verstärkung der Glukoseaufnahme durch BCR/ABL im Standardmedium bestätigt wurde, konnte im Minimal-medium Gegenteiliges gezeigt werden. Zudem wurde ein Unterschied im Aminosäurestoff-wechsel zwischen BaF3 + IL-3 und BaF3-BCR/ABL + IL-3 im Minimalmedium deutlich. Die therapeutische Relevanz des gezeigten Einflusses der Nährstoffrestriktion konnte anschlie-ßend in der Tyrosinkinaseinhibitortitration dargestellt werden, da die Medikamente in Abhän-gigkeit vom Medium unterschiedliche Wirkungen zeigen. Insgesamt bieten die Ergebnisse einen metabolischen Erklärungsansatz für das Überleben von Tumorstammzellen in nährstoffarmen Arealen des Knochenmarks unter Therapie und Raum für neue Therapieansätze.

3BP2 was originally identified through its interaction with the ABL kinase. Fusion of ABL with the BCR gene forms the BCR-ABL onco-protein, which is causative in Chronic Myeloid Leukemia (CML) and acute lymphoid leukemia (ALL). Due to the ability of 3BP2 to regulate ABL activity in osteoblasts, we hypothesize that 3BP2 modulates BCR-ABL signalling. Overexpression of 3BP2 in the CML-T1 cell line produced a marked decrease in global tyrosine phosphorylation. 3BP2 overexpression also resulted in a significant increase in CML-T1 cell growth, accompanied by altered ERK1/2, AKT, SYK, LYN, HCK, and CBL phosphorylation and expression. A phospho-SRC family protein and a 116 kDa phospho-protein were identified as 3BP2 interaction partners in response to BCR-ABL activation. BCR-ABL bone marrow transplantation (BMT) models in 3bp2-/- mice exhibit accelerated disease compared to wild-type mice, with altered leukemic phenotype. In conclusion, 3BP2 is able to modulate signalling through BCR-ABL and affect BCR-ABL-induced disease outcome.

3BP2 was originally identified through its interaction with the ABL kinase. Fusion of ABL with the BCR gene forms the BCR-ABL onco-protein, which is causative in Chronic Myeloid Leukemia (CML) and acute lymphoid leukemia (ALL). Due to the ability of 3BP2 to regulate ABL activity in osteoblasts, we hypothesize that 3BP2 modulates BCR-ABL signalling. Overexpression of 3BP2 in the CML-T1 cell line produced a marked decrease in global tyrosine phosphorylation. 3BP2 overexpression also resulted in a significant increase in CML-T1 cell growth, accompanied by altered ERK1/2, AKT, SYK, LYN, HCK, and CBL phosphorylation and expression. A phospho-SRC family protein and a 116 kDa phospho-protein were identified as 3BP2 interaction partners in response to BCR-ABL activation. BCR-ABL bone marrow transplantation (BMT) models in 3bp2-/- mice exhibit accelerated disease compared to wild-type mice, with altered leukemic phenotype. In conclusion, 3BP2 is able to modulate signalling through BCR-ABL and affect BCR-ABL-induced disease outcome.

The natural history of CML has been transformed in recent years by the introduction of Glivec[superscript TM] (imatinib mesylate), an ABL kinase inhibitor, which provides the new treatment paradigm for chronic phase CML. While the majority of patients with CP-CML respond very well to imatinib, there are approximately 15% of patients who fail to respond, or respond suboptimally. While the major cause of secondary imatinib resistance can be attributable to kinase domain mutations, the underlying cause of primary resistance is yet to be elucidated. Utilizing the phosphorylation of the adaptor protein Crkl, an immediate downstream partner of BCRABL, as a surrogate measure of BCR-ABL kinase activity, a large interpatient variation in the degree of imatinib induced kinase inhibition achieved in-vitro, was observed in previously untreated CP-CML patients. The observed in-vitro sensitivity was a good predictor of molecular response in patients treated with 600mg imatinib as front line therapy. Furthermore, analysis of the in-vivo reduction in p-Crkl mediated measured in blood cells in response to imatinib over the first 28 days of therapy, revealed that patients with higher % reductions respond significantly better over a two year period, than those with lower % reductions. Using 14-C labelled imatinib, it was demonstrated that this intrinsic sensitivity correlated to the amount of drug which was retained within the target haemopoietic cell, and furthermore, that a critical determinant of the active influx of imatinib, was the functional activity of the human organic cation transporter -1 (OCT-1), as determined by a prazosin (potent inhibitor of OCT-1) inhibition assay. Patients with high OCT-1 Activity had superior molecular responses when compared to those with low OCT-1 Activity, but in those patients who could tolerate increased imatinib dose, these inferior responses could be largely overcome. In contrast, Nilotinib, a more potent second generation tyrosine kinase inhibitor, is not dependent on OCT-1 for influx, making it a possible treatment choice for patients with low OCT-1 Activity. Both imatinib and nilotinib interact with the efflux transporters ABCB1, and ABCG2. In combination studies imatinib results in a significantly increased intracellular concentration of nilotinib, most likely through interaction with these efflux transporters. Furthermore, commonly used therapies such as proton pump inhibitors also interact with ABCB1 and ABCG2, and demonstrable changes in intracellular drug concentrations were observed in-vitro with concomitant administration of these agents and imatinib or nilotinib at clinically relevant concentrations. In conclusion, these data demonstrate that the degree of kinase inhibition mediated in-vitro and in-vivo by imatinib, is a critical determinant of subsequent molecular response. This intrinsic sensitivity to imatinib induced kinase inhibition is related to the activity of the OCT-1 protein. This protein is not involved in the transport of nilotinib, suggesting it as a possible treatment alternative in those patients with low OCT-1 Activity. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1319077 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

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Les protéines STAT5A et B sont des facteurs de transcription jouant un rôle important dans l'hématopoïèse normale et leucémique. Ce sont en effet des effecteurs essentiels d’oncogènes à activité tyrosines kinases, tels que BCR-ABL ou JAK2V617F responsables de la genèse d’hémopathies malignes. Ces oncogènes régulent la production de ROS (Reactive Oxygen Species) via l’activation de différentes voies de signalisation impliquées dans la prolifération et la survie cellulaire. Dans ce travail de thèse, nous montrons que l’activation oncogénique de STAT5, induite par BCR-ABL, favorise un stress oxydatif dans les cellules de Leucémie Myéloïde chronique (LMC), via la répression de l’expression des enzymes anti-oxydantes catalase et glutaredoxine-1 et la modulation potentielle de l’activité des NADPH oxydases. Nous montrons pour la première fois que l’effet pro-oxydant de STAT5 est régulé par la phosphorylation sur tyrosine de ces protéines et que les formes non phosphorylées et transcriptionnellement inactives exercent un effet anti-oxydant et protecteur contre le stress oxydatif, via des mécanismes non canoniques. Cette dualité de fonction est illustrée dans un modèle de co-culture de cellules de LMC et de cellules stromales médullaires, que nous avons développé dans le laboratoire afin de mimer le microenvironnement médullaire des cellules leucémiques. Dans ce modèle, nous montrons que le contact avec les cellules stromales permet d’inactiver STAT5 dans les cellules leucémiques et donc de promouvoir son activité anti-oxydante. Nous observons également un arrêt de prolifération et une entrée en phase G0 du cycle cellulaire des cellules leucémiques au contact des cellules stromales, ainsi qu’une résistance accrue de ces cellules à l’Imatinib, un inhibiteur de BCR-ABL. Ces données suggèrent un lien important entre activité anti-oxydante de STAT5, quiescence et chimio résistance des cellules leucémiques. / The Signal Transducer and Activator of Transcription factors 5A and B are two closely related STAT family members that play a major role in normal and leukemic hematopoiesis. STAT5 proteins are frequently activated in hematopoietic neoplasms and are targets of various tyrosine kinase oncogenes such as BCR-ABL and JAK2V617F. Both oncogenes were shown to stimulate the production of intracellular ROS (Reactive Oxygen Species) in leukemic cells and evidences for a cross talk between STAT5 and ROS metabolism have recently emerged. Herein, we demonstrate that sustained activation of STAT5 induced by BCR-ABL promotes ROS production in Chronic Myeloid Leukemia (CML) cells by repressing expression of two antioxidants, catalase and glutaredoxin1 and by possible functional interactions with NADPH oxidase complexes. We also provide compelling evidences that tyrosine phosphorylation regulate the pro-oxidant activity of STAT5 and that non phosphorylated STAT5 displays antioxidant properties and protection against oxidative stress via non-genomic effects. This dual function of STAT5 is also illustrated in an in vitro microenvironment model that we develop in our laboratory to analyze interactions between bone marrow stromal cells and CML cells. Using these coculture experiments, we show that STAT5 phosphorylation was reduced and its antioxidant activity enhanced in leukemic cells in contact with stromal cells. We also demonstrate in this model that leukemic cells stopped dividing, entered a quiescent G0 state and became resistant to Imatinib, a BCR-ABL kinase inhibitor. Collectively, these findings suggest an important link between antioxidant activity of STAT5, quiescence and resistance to chemotherapeutic agents in leukemic cells.

Die vorliegende Arbeit handelt von einer in vitro Untersuchung der Stoffwechselveränderun-gen durch die Expression von BCR/ABL bei BaF3-Zellen, einer murinen, IL-3-abhängigen B-Zelllinie. Die Zellen wurden in nährstoffreichem Standard- und in durch Titrationen ermittel-tem nährstoffarmem Minimalmedium auf unterschiedliche Stoffwechselaktivität in Abhän-gigkeit von BCR/ABL-Expression untersucht sowie auf die zusätzliche Beeinflussbarkeit durch IL-3. Danach wurden vergleichend zwischen den 2 Zelllinien (BaF3 und BaF3-BCR/ABL) im Minimalmedium und im Standardmedium Metabolite wie Glukose, Laktat und Aminosäuren bestimmt, wobei BaF3-BCR/ABL sowohl mit als auch ohne IL-3 kultiviert wur-de. Um den Einfluss von Nährstoffrestriktion auf die Therapie zu zeigen, wurden anschlie-ßend vergleichend in den beiden Medien die Tyrosinkinaseinhibitoren Imatinib und Nilotinib titriert. Die Ergebnisse zeigen, dass BaF3-BCR/ABL einen Wachstumsvorteil im Minimalmedium hat, welcher im Standardmedium nicht vorliegt. Während die bereits bekannte Verstärkung der Glukoseaufnahme durch BCR/ABL im Standardmedium bestätigt wurde, konnte im Minimal-medium Gegenteiliges gezeigt werden. Zudem wurde ein Unterschied im Aminosäurestoff-wechsel zwischen BaF3 + IL-3 und BaF3-BCR/ABL + IL-3 im Minimalmedium deutlich. Die therapeutische Relevanz des gezeigten Einflusses der Nährstoffrestriktion konnte anschlie-ßend in der Tyrosinkinaseinhibitortitration dargestellt werden, da die Medikamente in Abhän-gigkeit vom Medium unterschiedliche Wirkungen zeigen. Insgesamt bieten die Ergebnisse einen metabolischen Erklärungsansatz für das Überleben von Tumorstammzellen in nährstoffarmen Arealen des Knochenmarks unter Therapie und Raum für neue Therapieansätze.