Zelluläre Seneszenz beschreibt einen terminalen Zellzyklus-Arrest. Nach zellulärem Stress u. a. durch aktivierte Onkogene oder DNA-schädigende Chemotherapie wird Seneszenz induziert und kann so zur Tumorsuppression bzw. zum Behandlungserfolg beitragen. Vor kurzem wurde gezeigt, dass der Transkriptionsfaktor NF-kappaB – welcher bisher vor allem durch seine onkogenen Funktionen mit Krebs in Verbindung gebracht wurde - bei der Seneszenz-assoziierten Zytokinausschüttung mitwirkt und den seneszenzten Phänotyp möglicherweise sogar verstärkt, wodurch NF-kappaB potentiell eine tumorsuppressive Rolle zukäme. Ziel dieser Arbeit ist die Untersuchung des NF-kappaB-Signalweges in Seneszenz und Therapie. In der vorliegenden Arbeit zeige ich die deutliche Aktivierung von NF-kappaB nach Therapie-induzierter Seneszenz (therapy-induced senescence, TIS) und erhöhte Expression NF-kappaB-regulierter Zytokine. TIS ist vor allem in vivo mit starker Aktivität des NF-kappaB-Signalweges assoziiert und von selbiger abhängig. Primäre Eµ-myc-transgene Mauslymphome wurden nach ihrer endogenen NF-kappaB-Aktivität klassifiziert bzw. mit inhibierenden und aktivierenden NF-kappaB-Konstrukten modifiziert, welche auch in diffusen großzelligen B-Zell Lymphomen (diffuse large B-cell lymphoma, DLBCL) als natürlich vorkommende Mutationen gefunden wurden. Über einen neuartigen „Cross-Species“-Vergleich wurden Bcl2-hochexprimierende Keimzentrums-B-Zell-DLBCL (germinal center B-cell type, GCB) als klinisch relevante Gruppe identifiziert, welche nach NF-kappaB-Hyperaktivierung signifikant besser auf Therapie ansprach. Diese Ergebnisse zeigen eine kontextspezifische, d. h. von „onkogenen Netzwerken“ abhängige Rolle des NF-kappaB Signalweges unter Chemotherapie. Diese Information könnte für künftige klinische Studien bedeutsam sein, da sie Bedingungen aufzeigt, unter denen NF-kappaB als Vermittler einer erwünschten Therapie-induzierten Seneszenzantwort eher nicht inhibiert werden sollte. / Cellular senescence is a terminal cell-cycle arrest program that is executed in response to cellular stresses, such as activated oncogenes or DNA-damaging anti-cancer chemotherapy, where it serves as a tumor-suppressive mechanism or contributes to treatment outcome, respectively. Recently, transcription factor NF-kappaB which has long been linked to cancer development primarily through its oncogenic functions, has been postulated to participate in a senescence-associated and possibly senescence-reinforcing cytokine response, thereby suggesting a tumor-restraining role for NF-kappaB. The aim of my PhD project was to understand the role of the NF-kappaB pathway in senescence and cancer treatment outcome. In this thesis, I show markedly elevated NF-kappaB activity upon therapy-induced senescence (TIS), associated with strong upregulation of NF-kappaB-controlled cytokines. TIS is associated with and depends on hyper-activated NF-kappaB signaling. By characterization and genetic engineering of primary mouse lymphomas according to distinct NF-kappaB-related oncogenic networks reminiscent of diffuse large B-cell lymphoma (DLBCL) subtypes, Bcl2-overexpressing germinal center B-cell-like (GCB) DLBCL were identified as a clinically relevant subgroup with significantly superior outcome when NF-kappaB is hyperactive. These results demonstrate the context-dependent role of NF-kappaB signaling in cancer therapy and unveil oncogenic scenarios in which NF-kappaB hyperactivity unexpectedly accounts for superior long-term outcome to therapy. This finding has significant ramifications for future clinical trials that aim at inhibiting NF-kappaB activity based on the assumption of its detrimental impact on treatment outcome.

Bisher wurde davon ausgegangen, dass der IKK-komplex durch Regulation des Transkriptionsfaktors NF-kappaB die stressinduzierte Expression von Zielgenen steuert. Im Rahmen der hier vorgelegten Dissertation konnte jedoch gezeigt werden, dass der IKK-Komplex unabhängig von seiner Rolle in der NF-kappaB-Aktivierung die Stabilität einer Vielzahl von mRNAs kontrolliert. Mittels der Kombination von Ko-Immunopräzipitationsstudien und SILAC-MS konnte die induzierte Interaktion der regulatorischen Untereinheit des IKK-Komplexes IKKgamma mit dem Gerüstprotein EDC4 (Enhancer of Decapping 4) nachgewiesen werden. EDC4 ist eine essentielle Komponente sogenannter zytoplasmatischer „Processing Bodies“ (P-Bodies). Diese fungieren als Depots für die Speicherung von mRNAs, aber auch als Orte der mRNA-Degradation und der miRNA-vermittelten Repression spezifischer Zielgene. Die Interaktion von IKKgamma mit EDC4 konnte durch verschiedene Stimuli induziert werden. Dazu zählen DNA-Schäden durch Doppelstrangbrüche, aber auch die Aktivierung von Oberflächenrezeptoren durch TNFalpha und IL-1beta. EDC4 dient darüber hinaus als Substrat der Kinase IKKbeta. Mittels Massenspektrometrie und Kinaseassays konnten vier IKK-abhängige Phosphorylierungsstellen identifiziert werden. Die IKK-vermittelte Phosphorylierung von EDC4 ist essentiell für die Regulation von mRNAs und die damit verbundene Bildung der zytoplasmatischen P-Bodies. Diese Befunde konnten sowohl in stabilen induzierbaren Zelllinien, mittels transienter Transfektion und durch den Gebrauch von Kinaseinhibitoren in primären als auch in Krebszelllinien bestätigt werden. mRNA-Stabilitätsassays und eine RNA-Seq Analyse bestätigten die stressinduzierten Änderungen in den Halbwertszeiten spezifischer Transkripte und offenbarten einen gemeinsamen Regulationsmechanismus des IKK-Komplexes mit EDC4. / The IKK complex is deemed to regulate gene expression through the activation of the transcription factor NF-kappaB. Here I describe an NF-kappaB-independent function of the IKK complex in regulating mRNA stability across different cell types and stimuli. A SILAC-MS screen for interaction partners of the regulatory subunit IKKgamma revealed an inducible interaction with Enhancer of mRNA Decapping 4 (EDC4). EDC4 is an essential component of cytoplasmic processing bodies (P-bodies). P-bodies function as sites of mRNA storage, degradation and miRNA-mediated silencing. Interaction between IKKgamma and EDC4 can be induced by various stimuli, including DNA damage, TNFalpha and IL-1beta. EDC4 was identified as a novel IKK substrate and four IKKbeta phosphorylation sites were determined by mass spectrometry and in kinase assays. Stable inducible cell lines, transient transfection and kinase inhibitors were used in different human cancer and in primary cell lines and demonstrated that phosphorylation of EDC4 by IKK is essential for formation of P-Bodies in response to numerous stimuli. mRNA stability assays confirmed stress-induced changes in the half-life of target mRNAs and revealed common regulation of mRNA stability by IKK and EDC4. The transcriptome-wide reach of this joint regulation was assessed via RNA-Seq analysis.

Die Zellen des Hodgkin-Lymphoms sind, neben einer permanenten Aktivierung des NF-kB Signalweges, durch eine konstitutive AP-1 Aktivität gekennzeichnet. Die vorliegende Arbeit beschäftigte sich mit der Analyse der aberranten AP-1 Aktivität in Zellen des Hodgkin-Lymphoms. Von besonderem Interesse ist in diesem Zusammenhang der JUN Promotor, da c-Jun unter anderem die Fähigkeit besitzt seinen eigenen Promotor positiv zu regulieren (Autoregulation). Im Rahmen dieser Arbeit wurden Faktoren, die mit dem JUN Promotor in Zellen des Hodgkin-Lymphoms assoziiert sind, über verschiedene chromatographische Reinigungsschritte angereichert, mittels Massenspektrometrie identifiziert und hinsichtlich ihres Einflusses auf die c-Jun Expression analysiert. Dabei wurden die zwei Faktoren ATF-3, aus der Familie der AP-1 Proteine, und p52, aus der NF-kB Familie, hinsichtlich ihres Einflusses auf die Überexpression von c-Jun in Hodgkinzellen genauer untersucht. Die hier aufgeführten Ergebnisse tragen zu einem besseren Verständnis der Regulation der konstitutiven AP-1 Aktivität in den Hodgkinzellen bei. Im Rahmen dieser Arbeit konnten zwei Faktoren identifiziert werden, die maßgeblich an der Regulation der Expression von c-Jun in Hodgkinzellen beteiligt sind. Eine besondere Rolle kommt dabei dem Transkriptionsfaktor p52 zu, da dieser unter anderem auch die Expression anderer Mitglieder der AP-1 Familie reguliert. Ein weiterer Befund dieser Arbeit, dass p50 und p52 die zentralen Komponenten der konstitutiven NF-kB Aktivität sind, rückt p52 in das Zentrum zukünftiger Forschung. Die Befunde dieser Arbeit belegen, dass die aberrante Aktivierung von AP-1 im Hodgkin-Lymphom nicht auf ein einzelnes Ereignis in der Zelle zurückzuführen ist, sondern das Ergebnis eines komplexen Zusammenspiels vieler Faktoren ist. / The cells of Hodgkin''s lymphoma are characterized by a permanent activation of the NF-kB signaling pathway and a constitutive AP-1 activation. The present study focused on the analysis of aberrant AP-1 activity in cells of Hodgkin''s lymphoma. Of particular interest in this context is the JUN promoter, since c-Jun has the ability to regulate its own promoter (autoregulation). In this work different JUN promoter associated factors were enriched through various chromatographic purification steps, identified by Mass spectrometry and analyzed in terms of its influence on the expression of c-Jun. The focus was on specific factors in cells of Hodgkin''s lymphoma. The two factors ATF-3 and p52, were analyzed in terms of their influence on the overexpression of c-Jun in cells of Hodgkin''s lymphoma. Another interesting finding of this study is, p50 and p52 are central components of the constitutive NF-kB activity. This puts p52 in the center of future research. The results presented here contribute to a better understanding of the regulation of the constitutive AP-1 activity in the Hodgkin cells. In this work two Factors (ATF3 and p52) could be identified, which are are involved in the regulation of the expression of c-Jun in Hodgkin cells. A special role is played by the transcription factor p52, which also regulates the expression of other members of the AP-1 family. The findings of this study also show that the aberrant activation of AP-1 in Hodgkin''s lymphoma is not due to a single event in the cell. It is rather a result of a complex interplay of many factors.

Bakterielle Infektionen aktivieren den Transkriptionsfaktor NF-κB. Dies trägt sowohl zur Immunantwort, als auch zum Überleben der Wirtszellen bei. In Infektionen mit dem Bakterium Helicobacter pylori stellt diese doppelte Funktion einen mechanistischen Link zwischen der chronischen Entzündung und der Krebsentstehung dar. Für das intrazelluläre Bakterium Legionella pneumophila ist das Überleben der Wirtszelle notwendig für die Vermehrung. Bisher sind jedoch bei beiden Infektionen die Signalwege, die zur NF-κB-Aktivierung führen, nicht ausreichend untersucht. Um NF-κB analysieren zu können, wurden hier monoklonale Zelllinien hergestellt, die ein Fusionsprotein der NF-κB-Untereinheit p65 mit GFP stabil exprimieren. Die Kerntranslokation von p65-GFP kann mit Fluoreszenz-Mikroskopie visualisiert und mit automatischer Bildanalyse quantifiziert werden. Mit dieser Methode konnte zum ersten Mal eine durch ein Bakterium induzierte Oszillation von p65-GFP gezeigt werden. In einem RNAi-basierten Hochdurchsatzscreen wurde diese Methode eingesetzt um neue Faktoren im NF-κB-Signalweg zu identifizieren. Dabei wurde die Infektion mit H. pylori mit den Induktoren TNFα und IL-1β verglichen. Insgesamt wurden 24 Regulatoren identifiziert. Die Identifikation dieser Faktoren vertieft nicht nur unser Verständnis des NF-κB-Signalweges, sondern bietet auch neue molekulare Ziele für mögliche zukünftige Therapien. Bei der detaillierten Analyse der L. pneumophila-induzierten NF-κB-Aktivierung konnte ein einzigartiger, zwei-phasiger Ablauf gezeigt werden. Zunächst verursachte bakterielles Flagellin eine starke, aber kurze Aktivierung. Später war p65 dauerhaft über Stunden im Kern lokalisiert, was eng an die Replikation der Bakterien gekoppelt war. Da die kontinuierliche Kernlokalisation für Transkriptionsfaktoren der NF-κB Familie sehr ungewöhnlich ist, könnte dies ein Hinweis für eine Manipulation durch das Bakterium sein, um das Überleben der Wirtszelle zu sichern. / Infection with pathogens leads to activation of the transcription factor NF-κB. This contributes both to the immune response as well as survival of host cells. In infection with the bacterium Helicobacter pylori, this dual function provides a mechanistic link between the chronic inflammation and cancer development. In infection with the intracellular pathogen Legionella pneumophila, the survival oft he host cell is necessary for replication. However, in both infections, the signaling pathways leading to NF-κB activation are not well understood. Here, in order to investigate NF-κB activation, monoclonal cell lines were generated that stably express the NF-κB subunit p65 fused to GFP. Nuclear translocation of p65-GFP can be visualized by fluorescence microscopy and quantified by software-based picture analysis. Using this technology, oscillations of p65-GFP nuclear translocation could be shown after H. pylori infection. To identify new factors important for NF-κB activation, the new assay was used to conduct an RNAi-based screen. In the screen, infection with H. pylori was compared to induction with the cytokines TNFα and IL-1β. In total, 24 key regulators for NF-κB were identified. The identification of these factors broadens our understanding of NF-κB signaling and could provide targets for future therapies. Finally, detailed observation of NF-κB activation induced by L. pneumophila in single cells revealed a unique, biphasic NF-κB activation. During the first hours, bacterial flagellin induced strong but transient activation. Then, p65 translocated continuously to the nucleus over hours without oscillation. Testing an array of bacterial mutants, a tight link between bacterial replication and continuous NF-κB activation could be shown. Because this continuous nuclear localization is very unusual for a transcription factor of the NF-κB family, this indicates that L. pneumophila could manipulate NF-κB to ensure host cell survival.

Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, and dysregulation of caspase-9 splice variant ratio or expression of caspase-9b isoform has been linked to augmentation of the anchorage-independent growth and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. This study focuses on cell signaling pathway(s) regulating the alternative splicing of caspase-9 pre-mRNA and mechanistic roles of caspase-9b in a certain oncogenic/survival pathway. In regards to the former, we have identified hnRNP U as a novel splice-enhancer associated with exon 3 of caspase-9 (C9/E3). Moreover, hnRNP U binds specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interferes with hnRNP U for binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Overall, a mechanistic model has been revealed where hnRNP U competes with hnRNP L for C9/E3 binding to enhance the inclusion of the four-exon cassette, and this splice-enhancing effect is blocked by the AKT pathway via phosphorylation of hnRNP L. As to the latter aim, it is unknown about the mechanistic roles of caspase-9b besides the inhibitory effect on caspase-9a processing. In this study, caspase-9b has been demonstrated to have a dual function in regulating the survival/oncogenic nuclear factor κB (NF-κB) pathway, which is independent from modulating caspase-9a activation. In particular, caspase-9b has been shown to activate the canonical arm and inhibit the non-canonical arm of the NF-κB pathway by destabilizing NF-κB inhibitor alpha (IκB-α) and NF-κB-inducing kinase (NIK). Importantly, this new role for caspase-9b contributes to the enhanced survival and anchorage-independent growth of NSCLC cells conferred by caspase-9b expression. Further mechanistic studies have demonstrated a direct association of caspase-9b with the cellular inhibitor of apoptosis 1 (cIAP1), a regulatory factor in both arms of the NF-κB network, via its IAP-binding motif. Through this interaction, caspase-9b induces the E3 ligase activity of cIAP1, which regulates NF-κB activation, and promotes the survival, anchorage-independent growth and tumorigenicity of NSCLC cells. Overall, a novel tumorigenic mechanism has been identified, by which alternative mRNA processing regulates the NF-κB signaling independent of external agonist.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

NF-κB transcription factors are regulators of a wide spectrum of genes involved in immune responses and inflammation as well as cellular proliferation and survival. Transcriptionally competent NF-κB dimers are retained in the cytoplasm of resting cells by binding to inhibitors of NF-κB (IκBs). Stimuli that activate NF-κB converge on the activation of the IκB kinase (IKK), resulting in phosphorylation and subsequent proteasomal degradation of IκB. This releases functional NF-κB dimers that rapidly move to the nucleus where they regulate transcription of NF-κB-dependent target genes. The study of signalling to NF-κB from T and B lymphocyte antigen receptors is a field of intense investigation, and much attention is focused on the complex of the molecular scaffolding proteins Carma1, Bcl10 and MALT1. Together, these are crucial for the organisation of a structure beneath the activated receptor, termed the immunological synapse. IKK is recruited to this structure and becomes activated, subsequently leading to activation of NF-κB. Calcium (Ca2+) is a ubiquitous intracellular messenger that is involved in the regulation of numerous aspects of cellular function, including transcription. NF-κB activity is known to be regulated by changes in intracellular Ca2+ levels, such as those created by antigen receptor activation, but the mechanisms are to a large extent undefined. Ca2+ signals in cells are transmitted predominantly by the ubiquitous Ca2+ sensor protein calmodulin (CaM). Signalling that increases the intracellular Ca2+ concentration leads to binding of Ca2+ to CaM, which changes its structure, thereby allowing it to interact with a new range of target proteins. The studies of NF-κB signalling in lymphocytes presented here reveal that CaM is involved, both directly and indirectly, in the regulation of NF-κB. CaM was found to interact directly and in a Ca2+-dependent manner with the NF-κB proteins RelA and c-Rel after their signal-induced release from IκB. The interaction of CaM with c-Rel, but not RelA, was found to be inhibitory for its nuclear accumulation and transcriptional activity on Ca2+-regulated IL-2 and GM-CSF promoters; thus, CaM binding was found to differentially regulate c-Rel and RelA in lymphocytes. CaM was also shown to interact directly and in a Ca2+-dependent manner with Bcl10. The interaction was mapped to the Carma1-interacting CARD domain of Bcl10 and was found to have a negative effect on the ability of Bcl10 to bind to Carma1. Binding of CaM to Bcl10 also had a negative effect on activation of NF-κB after T cell receptor stimulation, since a point mutant of Bcl10 with reduced binding to CaM showed increased activation of an NF-κB reporter in Jurkat T cells, which was further enhanced by TCR-activating stimuli. In addition, CaM was found to positively regulate NF-κB activation indirectly through CaM-dependent kinase II (CaMKII). Inhibitors of CaM and CaMKII were shown to inhibit IκBα degradation in lymphocytes induced by phorbol ester or T cell receptor stimulation. The actions of CaMKII were mapped to a point upstream of IKK activation and further studies revealed that CaMKII is recruited to the immunological synapse, where it inducibly interacts with and phosphorylates Bcl10 at multiple sites. Phosphorylation of Bcl10 by CaMKII was shown to be important for the ability of Bcl10 to activate NF-κB, since mutation of the phosphorylation sites of Bcl10 inhibited Bcl10-induced transcriptional activity of NF-κB, in part by preventing signalinduced ubiquitination and degradation of Bcl10.

La transició epiteli-mesènquima (TEM) és un procés en què cèl lules epitelials, immòbils i amb polaritat apico-basal transiten cap un fenotip mesenquimal o fibroblàstic. L'expressió del factor de transcripció snail1 és suficient per induir TEM en cèl lules en cultiu i és necessari per la majoria de les TEM fisiològiques descrites. Snail1 és un membre de la família de proteïnes amb dits de Zinc que reprimeix gens epitelials (com l'E-cadherina) a través de la unió directa a seqüències especifiques dels promotors anomenades caixes E i posterior reclutament de corepressors. La TEM també es caracteritza per l'activació de gens mesenquimals, però el mecanisme pel qual snail1 indueix l'expressió d'aquests és poc conegut. En aquest treball demostrem que snail1 actua a nivell transcripcional per incrementar els nivells dels marcadors mesenquimals FN1 (fibronectina) i LEF1 (de l'anglès, lymphoid enhancer-binding factor 1) a través d'un mecanisme nou per aquesta proteïna de dits de Zn que no requereix ni caixes E ni unió directa a l'ADN. A més a més, mostrem que, per a dur a terme l'activació, snail1 coopera amb dos factors de transcripció ja descrits en relació a la TEM: beta-catenina i NF-kappa-B. Els nostres resultats també proven que l'expressió forçada de la E-cadherina evita aquesta cooperació i conseqüent activació gènica. A banda d'aquest mecanisme, també hem descrit que el factor de transcripció TFCP2c, que no havia estat prèviament relacionat amb TEM, és necessari per l'activació del gen FN1 induïda per snail1. / Epithelial-mesenchymal transition (EMT) is a cellular process by which no motile epithelial, apico-basal-polarized cells transit towards a motile mesenchymal front-backpolarized phenotype. Expression of the transcription factor snail1 is sufficient to induce EMT in cultured cells and it is required for most of the physiological EMTs described. Snail1 is a member of the Zn finger protein family that represses epithelial genes (such as E-cadherin) by directly binding to specific promoter sequences called E-boxes and subsequent recruitment of corepressors. EMT is also accompanied by activation of mesenchymal genes, however, little is known of how snail1 induces their expression.In this work we provide evidence that snail1 acts at the transcriptional level to increase the levels of the mesenchymal FN1 (fibronectin) and LEF1 (lymphoid enhancer-binding factor 1) genes through a novel mechanism for this Zn finger protein that does not require neither E-boxes nor direct binding to DNA. Furthermore, we describe a cooperative action in such mechanism between snail1 and two transcription factors previously related to EMT: beta-catenin and NF-kappaB. Our results also show that restoration of E-cadherin levels prevents such cooperation and subsequent activation. In addition, we also demonstrate that TFCP2c, which had not been previously linked to EMT, is also required for snail1-induced transcriptional activation of the FN1 gene.

Thesis (Ph. D.)--Indiana University, 2008. / Title from screen (viewed June 24, 2009). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Maureen Harrington. Includes vita. Non-Latin script record. Includes bibliographical references (leaves 126-132).

Glioblastoma Multiforme (GBM) is the most common lethal brain tumour associated with dismal survival rate. GBM is considered to be an incurable malignancy as these tumours evade all intricate attempts of therapy and no contemporary chemotherapeutic regimen is effective. Although the existence of cancer stem cells (CSCs) is still debatable, it is widely accepted that GBM has a small population of cells expressing CSC markers (~1%) that are highly resistant to chemo-radiation therapy. Recent evidence indicates that hypoxia induces cancer stem cell (CSC) phenotypes via epithelial-to-mesenchymal transition (EMT) that promote therapeutic resistance in solid tumours. Given that GBMs are extensively hypooxygenated heterogenous tumours, understanding the molecular relationship between hypoxia, biology of CSCs, EMT and chemoresistance would be invaluable for development of drugs that can target CSCs. Evidence suggests that hypoxia inducible factors (HIFs), NF-B and aldehyde dehydrogenase (ALDH) together orchestrate the stemness and chemoresistance in hypoxia induced CSCs. But the insights on the mechanisms still remain obscure. In this study we used an in vitro GBM CSC and hypoxia model along with NF-B-p65 and HIF transfected GBM cell lines to investigate the relationship between HIFs, NF-B activation and ALDH activity and their role in chemoresistance. The findings of this study demonstrated that GBM cells grown as spheres consist of a vast proportion of hypoxic cells with elevated CSC and EMT markers suggesting hypoxia induced EMT. GBM-CSCs are chemoresistant and displayed increased levels of HIFs, NF-B and ALDH activity. It was also observed that stable transfection of GBM cells with NF-B-p65 or HIFs induced CSC and EMT markers indicating their essential role in maintaining CSC phenotypes. The study also highlighted the importance of NF-B and ALDH in driving chemoresistance and the potential role of NF-B as the master regulator of hypoxia induced stemness in GBM cells. In this study, we used Disulfiram (DS), an anti-alcoholism drug, in combination with copper (Cu) to target the hypoxia-NF-B axis and inhibit ALDH activity to reverse chemoresistance in GBM CSCs. We showed that DS/Cu is cytotoxic to GBM cells and completely eradicated the resistant CSC population at low nanomolar levels in vitro. We also demonstrated that DS/Cu effectively inhibited GBM in vivo using newly formulated PLGA-DS nanoparticles. DS is an FDA approved drug with low/no toxicity to normal tissues and can freely pass through the blood brain barrier (BBB). Further study may lead to quick translation of DS into clinical trials.