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To overcome poor treatment response of pediatric high-risk acute lymphoblastic leukemia (ALL), novel treatment strategies are required to reactivate programmed cell death in this malignancy. Therefore, we take advantage of using small-molecule antagonists of Inhibitor of apoptosis (IAP) proteins, so called Smac mimetics such as BV6, which are described to overcome apoptosis resistance and thereby sensitize tumor cells for several apoptotic stimuli. To address the question whether redox alterations can sensitize leukemic cells for Smac mimetic-mediated cell death, we interfered with the cellular redox status in different ALL cell lines. Here, we show for the first time that redox alterations, mediated by the glutathione depleting agent Buthioninesulfoximine (BSO), prime ALL cells for BV6-induced apoptosis. Besides ALL cell lines, BV6/BSO cotreatment similarly synergizes in cell death induction in patient-derived primary leukemic samples. In contrast, the combination treatment does not exert any cytotoxicity against peripheral blood lymphocytes (PBLs) or mesenchymal stroma cells (MSCs) from healthy donors, suggesting some tumor selectivity of this treatment. We also identify the underlying molecular mechanism of the novel synergistic drug interaction of BSO and BV6. We demonstrate that both agents act in concert to increase reactive oxygen species (ROS) production, lipid peroxidation and finally apoptotic cell death. Enhanced ROS levels in the combination treatment account for cell death induction, since several ROS scavengers, like NAC, MnTBAP and Trolox attenuate BSO/BV6-induced apoptosis. BSO/BV6-induced ROS can be mainly classified as lipid peroxides, since the vitamin E derivate α-Tocopherol as well as Glutathione peroxidase 4 (GPX4), which both specifically reduce lipid-membrane peroxides, prevent lipid peroxidation, caspase activation and cell death induction. Vice versa, GPX4 knockdown and pharmacological inhibition of GPX4 by RSL3 or Erastin enhance BV6-induced cell death. Importantly, cell death induction critically depends on the formation of a complex consisting of RIP1/FADD/Caspase-8, since all complex components are required for ROS production, lipid peroxidation and cell death induction. Taken together, we demonstrate that BSO and BV6 cooperate to induce ROS production and lipid peroxidation which are eventually required for caspase activation and cell death execution. Collectively, findings of this study indicate that BV6-induced apoptosis is mediated via redox alterations offering promising new treatment strategy to overcome apoptosis resistance in ALL.
Recently, two of the most common types of bone cancers in children and young adults have been proven to exhibit vulnerability to poly(ADP)-ribose polymerase, (PARP) inhibitors (e.g. olaparib, talazoparib). Ewing’s sarcoma (ES) are reported to harbor a fusion gene EWS-FLI1 (85%), inducing tumorigenesis. Additional, as the fusion gene acts as aberrant transcription factor, it similarly induces elevated PARP expression levels sensitizing ES to PARP inhibition. Second, by an exome sequencing approach in a set of primary osteosarcomas (OS) we identified mutation signatures being reminiscent of BRCA deficiency. Therefore, the sensitivity of a panel of OS cell lines to either talazoparib single treatment or in combination with several chemotherapeutic drugs was investigated.
To screen ES tumor cell lines against PARP inhibitors we applied four different PARP inhibitors (talazoparib, olaparib, niraparib and veliparib) that are frequently being used for clinical studies. We combined those PARP inhibitors with a set of chemotherapeutics (temozolomide (TMZ), SN-38, etoposide, ifosfamide, doxorubicin, vincristine and actinomycin D) that are part of the first-line therapy of ES patients. Here, we demonstrate how PARP inhibitors synergize with TMZ or SN-38 to induce apoptosis, whereas the combination of PARP inhibitors with the other drugs are not favorable. By investigation of key checkpoints in the molecular mechanisms of cell death, the pivotal role of the mitochondrial pathway of apoptosis mediating the synergy between olaparib and TMZ was revealed.
Employing talazoparib monotherapy in combination with or without several chemotherapeutic drugs (TMZ, SN-38, cisplatin, doxorubicin, methotrexate and etoposide/carboplatin), the correlation between homologous recombination (HR) repair deficiency (BRCAness) and the response to talazoparib as prototypical PARP inhibitor was validated in different OS cell lines. By calculation of combination indices (CI) and fraction affected (Fa) values, we identified TMZ as the most potent chemotherapeutic drug in combination with talazoparib inducing the mitochondrial apoptotic pathway in OS.
In our studies of two independent tumor entities with contrary genetic background we identified the combination of PARP inhibitor and TMZ as being most effective. Our studies point out that after TMZ induced DNA methylation and concomitant PARP trapping, DNA damage-imposed checkpoint kinase activation consequently induces G2-cell cycle arrest. Subsequent, PARP inhibitor/TMZ causes MCL-1 degradation, followed by activation of BAK and BAX, succeeding in loss of mitochondrial outer membrane potential (LMMP) and activation of downstream effector-caspases in mitochondrial apoptosis. Our findings emphasize the importance of PARP inhibition in order to chemosensitize ES, which express high PARP levels, or OS that bear features of BRCAness.
HDAC inhibitors (HDACI), a new class of anticancer agents, induce apoptosis in many cancer entities. JNJ-26481585 is a second generation class І HDACI that displays improved efficacy in preclinical studies compared to the established HDACI SAHA (Vorinostat). Therefore, this study aims at evaluating the effects of JNJ-26481585 on human rhabdomyosarcoma (RMS) and at identifying novel synergistic interactions of JNJ-26481585 or the more common HDACI SAHA with different anticancer drugs in RMS cells. Indeed, we show that JNJ-26481585 and SAHA significantly increase chemotherapeutic drug-induced apoptosis in embryonal and alveolar RMS cell lines, when used in combination with chemotherapeutic agents (i.e. doxorubicin, etoposide, vincristine, and cyclophosphamide) which are currently used in the clinic for the treatment of RMS.
We demonstrate that JNJ-26481585 as single agent and in combination with doxorubicin induces apoptosis, which is characterized by activation of the caspase cascade, PARP cleavage, and DNA fragmentation. Induction of caspase-dependent apoptotic cell death is confirmed by the use of the broad-range caspase inhibitor zVAD.fmk, which significantly decreases both JNJ-26481585-triggered and combination treatment-mediated DNA fragmentation, and in addition completely abrogates loss of cell viability. Importantly, JNJ-26481585 significantly inhibits tumor growth in vivo in two preclinical RMS models, i.e. the chicken chorioallantoic membrane (CAM) model and a xenograft mouse model, supporting the notion that JNJ-26481585 hampers tumor maintenance. Also, in combination with doxorubicin JNJ-26481585 significantly reduces tumor growth in in vivo experiments using the CAM model.
Mechanistically, we identify that JNJ-26481585-induced apoptosis is mediated via the intrinsic apoptotic pathway, since we observe increased loss of mitochondrial membrane potential and activation of the proapoptotic Bcl-2 family members Bax and Bak. Interestingly, we find that JNJ-26481585 triggers induction of Bim, Bmf, Puma, and Noxa on mRNA level as well as on protein level, pointing to an altered transcription of BH3-only proteins as important event for the Bax/Bak-mediated loss of mitochondrial membrane potential as well as mitochondrial apoptosis induction upon JNJ-26481585 treatment. JNJ-26481585-initiated activation of Bax and Bak is not prevented with the addition of zVAD.fmk, suggesting that JNJ-26481585 first disrupts the mitochondria and subsequently activates the caspase cascade. When JNJ-26481585 is used in combination with doxorubicin, we observe not only an increase of proapoptotic Bcl-2 proteins, but also a decrease in the level of the antiapoptotic mitochondrial proteins Bcl-2, Mcl-1, and Bcl-xL. This indicates that Bax, Bak, Bim, and Noxa are crucial for JNJ-26481585-induced as well as JNJ/Dox treatment-induced apoptosis, since RNAi mediated silencing of Bax, Bak, Bim, and Noxa significantly impedes DNA fragmentation upon those treatments.
Furthermore, ectopic overexpression of Bcl-2 profoundly impairs both JNJ-26481585 and combination treatment-mediated apoptosis, abrogates caspase cleavage, and reduces activation of Bax and Bak, underlining the hypothesis that JNJ-26481585 initially targets the mitochondria and then activates caspases.
With the more commonly used HDACI SAHA we confirm the results obtained with the HDACI JNJ-26481585, since combination treatment with SAHA and doxorubicin also induces intrinsic apoptosis, which can be significantly diminished by zVAD.fmk or ectopic overexpression of Bcl-2. Treatment with SAHA and doxorubicin also affects expression levels of pro- and antiapoptotic mitochondrial proteins, thus shifting the balance towards the proapoptotic mitochondrial machinery, resulting in Bax/Bak activation, caspase activation, and subsequently apoptosis.
Taken together, we provide evidence that the HDACIs JNJ-26481585 and SAHA are promising therapeutic agents for the treatment of RMS and that combination regimens with HDACIs represent an efficient strategy to prime RMS cells for chemotherapy-induced apoptosis. These findings have important implications for mitochondrial apoptosis-targeted therapies of RMS.
Rhabdomyosarcoma (RMS) is the most frequent pediatric soft-tissue sarcoma comprising two major subtypes – the alveolar and the embryonal rhabdomyosarcoma. The current therapeutic regime is multimodal including surgery, radiation and chemotherapy with cytostatic drugs. Although the prognosis for RMS patients has steadily improved to a 5-year overall survival rate of 70% for ERMS and 50% for ARMS, prognosis for subgroups with primary metastases or relapsed patients is still less than 25%, highlighting the need for development of new therapies for these subgroups. Since cancer cells are addicted to their cancer promoting transcriptional program, remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as compelling anticancer strategy. However, in many cancer types BET inhibition was proved cytostatic but not cytotoxic emphasizing the need for combination protocols.
In this study we identify a novel synergistic interaction of the BET inhibitor JQ1 with p110α-isoform-specific Phosphoinositid-3-Kinase (PI3K) inhibitor BYL719 (Alpelisib) to induce mitochondrial apoptosis and global reallocation of BRD4 to chromatin. At first, we showed that JQ1 single treatment had cytostatic effects at nanomolar concentrations and inhibited MYC and Hedgehog (Hh) signaling in RMS known to promote proliferation of RMS. However, JQ1 single treatment barely induced cell death in RMS cells even at concentrations of up to 20 µM (< 20% cell death). Thus, we next tested combination approaches to elicit cell death. Since we previously identified synergistic cell death induction of Hh inhibition and PI3K inhibition in RMS cells we tested JQ1 in combination with the pan-PI3K/mTOR inhibitor PI-103 and the p110α-isoform-specific PI3K inhibitor BYL719. In addition, we tested JQ1 in combination with distinct HDAC inhibitors namely JNJ-26481585, SAHA (Vorinostat), MS-275 (Entinostat) and LBH-589 (Panobinostat) since the synergistic interaction of BET and HDAC inhibition has previously been described for other tumor entities.
Interestingly the synergism of cell death induction of JQ1/BYL719 co-treatment is superior to the synergism of JQ1 with pan-PI3K/mTOR inhibitor PI-103 or the tested HDAC inhibitors as confirmed by calculation of combination index. To investigate the molecular mechanisms underlying the synergy of JQ1/BYL719 co-treatment, we performed RNA-Seq and BRD4 ChIP-Seq experiments. RNA-Seq exhibited, that JQ1/BYL719 co-treatment shifted the overall balance of BCL-2 family gene expression towards apoptosis and increased gene expression of proapoptotic BMF, BCL2L11 (BIM) and PMAIP1 (NOXA) while decreasing gene expression of antiapoptotic BCL2L1 (BCL xL). These changes were verified by qRT-PCR and Western blot. Notably, BRD4 is phosphorylated upon JQ1/BYL719 co-treatment and globally reallocates BRD4 to chromatin. This BRD4 reallocation includes enrichment of BRD4 at the super-enhancer site of BMF, at the super-enhancer, typical enhancer and promoter regions of BCL2L11 (BIM) and at the PMAIP1 (NOXA) promoter, while JQ1 alone, as expected, reduces global chromatin binding of BRD4. Integration of RNA-Seq and BRD4 ChIP-Seq data underlines the transcriptional relevance of reallocated BRD4 upon JQ1/BYL719 co-treatment. Immunopreciptation studies showed, that RMS cells are initially primed to undergo mitochondrial apoptosis since BIM is constitutively bound to antiapoptotic BCL-2, BCL xL and MCL-1. JQ1/BYL719 co-treatment increased BIM expression and its neutralization of antiapoptotic BCL-2, BCL-xL and MCL-1 thereby rebalancing the ratio of pro- and antiapoptotic BCL-2 proteins in favor of apoptosis. This promotes activation of BAK and BAX resulting in caspase-dependent apoptosis. The functional relevance of proapoptotic re-balancing for the execution of JQ1/BYL719-mediated apoptosis was confirmed by individual silencing of BMF, BIM, NOXA or overexpression of BCL-2 or MCL-1, which all significantly rescued JQ1/BYL719-induced cell death. Execution of cell death by mitochondrial caspase-dependent apoptosis was veryfied by individual knockdown of BAK and BAX or caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk), which all significantly rescued JQ1/BYL719-induced cell death.
In summary, combined BET and PI3Kα inhibition cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins accompanied by reallocation of BRD4 to transcriptional regulatory elements of BH3-only proteins.
Acute lymphoblastic leukemia (ALL), a neoplastic disorder of blood cells of the lymphoid lineage, is the most frequent childhood cancer. In spite of increasing survival rates, the outcome for adults, infants or relapsed patients is still less favorable, highlighting the need for novel treatment options. Reactive oxygen species (ROS) are important signaling molecules that are involved in a variety of cellular pathways. As high ROS levels lead to oxidative stress and irreversible oxidation of cellular macromolecules, the production and elimination of ROS is tightly controlled. Therefore, cells express several antioxidant molecules and enzymes, including glutathione, catalase and the thioredoxin (Trx) system, to balance ROS levels. As cancer cells were found to have increased ROS levels that could contribute to tumor progression and metastasis, they rely strongly on these antioxidant systems to prevent oxidative damage, making cancer cells especially vulnerable to ROS-inducing treatments. ROS and oxidative stress have been shown to induce programmed cell death via different pathways, however the exact mechanisms that couples oxidative signaling and cell death is not completely understood.
As a disturbance of the cellular redox homeostasis was reported during leukemia development and progression, we wanted to determine the potential of Trx inhibitors for ALL therapy. Additionally, we aimed to further understand the role of ROS and subsequent protein oxidation in the induction and execution of programmed cell death.
First, we demonstrated that the Trx1 inhibitor PX-12 induced cell death in three ALL cell lines. Further analysis of the events leading to PX-12-induced cell death in FADD-deficient (FD) Jurkat cells revealed an increase in ROS levels and oxidation-mediated dimer formation of peroxiredoxin 3 (PRDX3). Interestingly cell death was inhibited by the thiol-containing antioxidant N-acetylcysteine (NAC), but not by non-thiol-containing ROS scavengers. PX-12 treatment further induced cleavage of caspase-9 and -3 and activation of the pro-apoptotic BCL-2 protein BAK, leading us to the conclusion that mitochondria-dependent apoptosis was induced. Interestingly, we could demonstrate an important role for the BH3-only protein NOXA in the mediation of PX-12-induced apoptosis as knock-down of NOXA prevented cell death induction and BAK activation. Our findings give novel insights into the mechanism of PX-12-induced cell death in ALL cell lines and underscores the potential of PX-12 for the treatment of ALL.
To further understand the processes leading to cell death upon inhibition of the Trx system, we analyzed global protein oxidation in Jurkat FD cells upon treatment with the Trx reductase inhibitor Auranofin. In line with previous results, Auranofin induced intrinsic apoptosis that was dependent on BAK and accompanied by increased ROS levels. Using a BIAM Switch Assay followed by mass spectrometry, we demonstrated that Auranofin treatment induced oxidation of over 200 proteins. We identified several proteins whose oxidation upon Auranofin treatment was expected, like Trx1, Trx2 and several peroxiredoxins. Additionally, we verified oxidation of APAF1-interacting protein (APIP) and protein arginine N-methyltransferase (PRMT1) that are both implicated in the regulation of apoptosis. With this analysis we were able to demonstrate that Auranofin treatment leads to changes in global protein oxidation. Whether oxidation of the determined proteins changes their functionality and contributes to apoptosis induction remains to be elucidated.
As we identified BAK as an important player in PX-12- and Auranofin-induced cell death in the previous parts of this study, we wanted to further understand its involvement in ROS-mediated cell death. First analyses in wild-type (WT) and BAK-/- murine embryonic fibroblasts (MEFs) revealed that BAK was essential for Auranofin-induced cell death and that this cell death was caspase-independent in MEFs. Interestingly, BAK oxidation was induced upon treatment with Auranofin, but not upon stimulation with the apoptosis-inducing compound Etoposide. Expression of mutated BAK, with either one or both oxidation-sensitive cysteines mutated to oxidation-insensitive serines, revealed that mutating already one cysteine protected cells from Auranofin , but not Etoposide-induced cell death. Of note, mutation of the BAK BH3 domain rescued MEFs from both, Auranofin- and Etoposide-mediated cell death. The presence of cysteine residues also altered BAK interactions as observed by a mass spectrometric analysis of Auranofin-treated MEFs expressing either WT or cysteine-less BAK. We identified interactions of WT BAK with proteins involved in mitochondrial fission and vesicle transport upon Auranofin treatment. Of note, interaction with proteins involved in apoptosis, like BAX or BCL-XL, was not changed between WT and cysteine-less BAK. Our results demonstrate a critical role for BAK oxidation in Auranofin-induced cell death. Furthermore, we identified novel oxidation-dependent BAK interaction partners.
To conclude, this study highlights the potential of ROS-inducing treatments for ALL therapy and provides novel insights into the redox regulation of programmed cell death.
Ubiquitination is regarded as one of the key post-translational modifications in nearly all biological processes, endowed with numerous layers of complexity. Deubiquitinating enzymes (DUBs) dynamically counterbalance ubiquitination events by deconjugating ubiquitin signals from substrates. Dysregulation of the ubiquitin code and its negative regulators drive various pathologies, such as neurological disorders and cancer.
The DUB ubiquitin-specific peptidase 22 (USP22) is well-known for its essential role in the human Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, mediating the removal of monoubiquitination events from Histone 2A and 2B (H2A and -B), thereby regulating gene transcription. In cancer, USP22 was initially described as a part of an 11-gene expression signature profile, predicting tumor metastasis, reoccurrence and death after therapy in a wide range of tumor cells. However, novel roles for USP22 have emerged recently, accrediting USP22 essential roles in regulating tumor development as well as apoptotic cell death signaling.
One of the hallmarks of cancer is the evasion of cell death, especially apoptosis, a form of programmed cell death (PCD). Necroptosis, a regulated form of necrosis, is regarded as an attractive therapeutic strategy to overcome apoptosis-resistance in tumor cells, although a profound understanding of the exact signaling cascade still remains elusive. Nevertheless, several ubiquitination and deubiquitination events are described in fine-tuning necroptotic signaling.
In this study, we describe a novel role for USP22 in regulating necroptotic cell death signaling in human tumor cell lines. USP22 depletion significantly delayed TNFa/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFa-induced nuclear factor-kappa B (NF-KB) signaling or TNFa-mediated extrinsic apoptosis. Intriguingly, re-expression of USP22 wildtype in the USP22 knockout background could re-sensitize HT-29 cells to TBZ-induced necroptosis, whereas re-constitution with the catalytic inactive mutant USP22 Cys185Ser did not rescue susceptibility to TBZ-induced necroptosis, confirming the USP22 DUB-function a pivotal role in regulating necroptotic cell death. USP22 depletion facilitated ubiquitination and unexpectedly also phosphorylation of Receptor-interacting protein kinase 3 (RIPK3) during necroptosis induction, as shown by Tandem Ubiquitin Binding Entities (TUBE) pulldowns and in vivo (de)ubiquitination immunoprecipitations. To substantiate our findings, we performed mass-spectrometric ubiquitin remnant profiling and identified the three novel USP22-regulated RIPK3 ubiquitination sites Lysine (K) 42, K351 and K518 upon TBZ-induced necroptosis. Further assessment of these ubiquitination sites unraveled, that mutation of K518 in RIPK3 reduced necroptosis-associated RIPK3 ubiquitination and additionally affected RIPK3 phosphorylation upon necroptosis induction. At the same time, genetic knock-in of RIPK3 K518R sensitizes tumor cells to TNFa-induced necroptotic cell death and amplified necrosome formation.
In summary we identified USP22 as a new regulator of TBZ-induced necroptosis in various human tumor cell lines and further unraveled the distinctive role of DUBs and (de)ubiquitination events in controlling programmed cell death signaling.
Inhibitoren der Apoptose (IAP, inhibitor of apoptosis) Proteine spielen eine wichtige Rolle in Bezug auf Zelltodregulation und es ist anzunehmen, dass eine Dysregulation dieser Proteine zu einer Tumorentwicklung und Tumorprogression beiträgt. Erhöhte Expressionslevel von IAP Proteinen verhindern die Aktivierbarkeit des Zelltodprogrammes von Tumorzellen und eine Reihe von Studien konnte bereits erhöhte IAP Level in Tumorzelllinien sowie in primären Tumorproben nachweisen. Des Weiteren korrelieren erhöhte Expressionslevel von IAPs in Tumoren mit Behandlungsresistenzen und schlechten Prognosen für die Patienten.
Das diffuse großzellige B-Zell Lymphom (DLBCL, diffuse large B-cell lymphoma) zählt zu den häufigsten Subtypen der Non-Hodgkin Lymphome (NHL) mit 40 % aller neu diagnostizierten NHL Fälle. DLBCL ist eine sehr heterogene Erkrankung die in drei verschiedene Gruppen klassifiziert wurde: aktivierter B-Zell Typ (ABC, activated B-cell), Keimzentrum B-Zell Typ (GCB, germinal center B-Cell) und Mediastinaler großzelliger B-Zell Typ (PMBL, primary mediastinal B-cell lymphoma). Erhöhte Expressionslevel von zellulärem IAP1 (cIAP, cellular IAP) und cIAP2 wurden ebenfalls in primären Tumorproben von DLBCL Patienten nachgewiesen. Smac mimetics wurden entwickelt, um IAPs zu antagonisieren und stellen damit eine Behandlungsstrategie für DLBCL Patienten dar, denn ca. 40 % aller DLBCL Patienten entwickeln ein Rezidiv oder erreichen gar keine Remission unter Standardtherapie. Jedoch ist der Effekt von Smac mimetics in einer Einzelbehandlung limitiert, weswegen Kombinationstherapien mit Smac mimetics eine vielversprechende Strategie für ihren klinischen Einsatz darstellen. Aus diesem Grund haben wir in dieser Arbeit den Effekt von Smac mimetic in Kombination mit Proteasom-Inhibitoren analysiert und einen speziellen Fokus auf den molekularen Mechanismus des ausgelösten Zelltodsignalweges gelegt.
Die Kombination verschiedener Konzentrationen des Smac mimetics BV6 mit dem Proteasom-Inhibitor carfilzomib (CFZ) löst in allen drei getesteten DLBCL Subtypen (ABC, GCB und PMBL) Zelltod aus. Die Kalkulation des Kombinationsindexes (CI, combination index) sowie des Bliss Scores, zwei quantitative Parameter zur Bestimmung eines Synergismus, zeigen, dass fast alle getesteten Kombinationen einen Synergismus aufweisen. Dies verdeutlicht, dass eine Co-Behandlung von BV6 und CFZ eine wirksame Kombination ist um Zelltod in DLBCL Zelllinien auszulösen. Außerdem zeigt eine Kombination von BV6 mit anderen Proteasom-Inhibitoren wie ixazomib (IXA) oder oprozomib (OPR), ebenfalls eine synergistische Reduktion der Zellviabilität. Diese Ergebnisse deuten darauf hin, dass der detektierte Effekt nicht auf eine Substanz limitiert ist, sondern, dass ein genereller Effekt von Smac mimetic und Proteasom-Inhibitoren vorliegt, um Zellviabilität in DLBCL zu reduzieren. BV6 und CFZ induzieren einen apoptotischen Zelltod, da sie die Spaltung und Aktivierung von Initiator- und Effektorcaspasen (Caspasen-3, -7, -8 und -9) initiieren und sich der induzierte Zelltod mit Hilfe des Caspasen-Inhibitors zVAD.fmk verhindern lässt. Die Behandlung mit BV6 und CFZ führt zu einer Akkumulation von NIK, ein Protein welches zur Aktivierung des non-kanonischen NF-kB Signalweges benötigt wird. Weitere Untersuchungen zeigen jedoch, dass NIK nicht an der Zelltodinduktion beteiligt ist, da eine siRNA-basierte Herunterregulierung des NIK Proteins keinen Einfluss auf die Zelltodinduktion nimmt. Ebenfalls ist der Zelltod unabhängig von dem TNFa Signalweg, da weder eine Behandlung mit dem TNFa Inhibitor Enbrel den Zelltod verringern kann noch eine zusätzliche Gabe von TNFa den Zelltod erhöht. Weitere mechanistische Studien zeigen eine kritische Rolle der mitochondrialen Apoptose für den BV6/CFZ-vermittelten Zelltod. Unter Behandlung mit BV6/CFZ wurde eine Aktivierung von BAX und BAK nachgewiesen, welche beide mit verantwortlich für die Porenbildung in der mitochondrialen Membran sind. Eine Herunterregulation dieser beiden Proteine mittels siRNA reduziert signifikant den durch BV6/CFZ-induzierten Zelltod auf ein Minimum. Gleichzeitig löst eine Co-Behandlung mit BV6/CFZ einen Verlust des mitochondrialen Membranpotentials (LOMMP, loss of mitochondrial membrane potential) aus. In Übereinstimmung mit den vorherigen Experimenten, zeigen wir eine Akkumulation von mitochondrialen reaktiven Sauerstoffspezies (ROS; reactive oxygen species), sowie einen generellen Anstieg des allgemeinen ROS Levels. Eine Behandlung mit BV6/CFZ zeigt eine deutliche Akkumulation des pro-apoptotischen Proteins NOXA. Um dessen funktionelle Relevanz zu überprüfen, wurde die Proteinmenge von NOXA mittels siRNA stark reduziert. Eine Behandlung mit der Kombination aus BV6 und CFZ zeigt daraufhin eine signifikant reduzierte Zelltodinduktion, was die funktionelle Relevanz von NOXA für den BV6/CFZ-vermittelten Zelltod unterstreicht. Immunopräzipitationsstudien zeigen, dass in RIVA und U2932 Zellen NOXA konstitutiv an seinen anti-apoptotischen Bindungspartner MCL-1 gebunden ist, was die Zellen bereits darauf vorbereitet Apoptose zu durchlaufen. Dieses sogenannte „primen“ für Apoptose wird durch die Behandlung mit BV6 und CFZ weiter verstärkt, da es die Bindung zwischen NOXA und MCL-1 weiter erhöht. Dadurch wird die Balance zwischen pro- und anti-apoptotischen Proteinen zu Gunsten der pro-apoptotischen Proteine verschoben und die Induktion von Apoptose begünstigt.
Insgesamt zeigen die Ergebnisse, dass DLBCL Zelllinien sensitiv auf eine Behandlung mit Smac mimetic und Proteasom-Inhibitor reagieren und damit eine mögliche neue Behandlungsstrategie für diese heterogene Tumorerkrankung darstellt.
Reactive oxygen species (ROS) are involved in various signalling mechanisms. Redox homeostasis is important in cancer cells, since they are dependent on upregulated antioxidant defence pathways to cope with elevated ROS levels. Therefore, targeting the antioxidant defence system and/ or increasing ROS to a lethal level may be a feasible strategy to counteract cancer cell progression.
Acute lymphoblastic leukaemia (ALL) is the most frequent malignant childhood cancer, displaying on one side resistance to cell death induction and on the other side elevated ROS levels. Therefore, inducing ferroptosis, a ROS- and iron-dependent cell death pathway might be useful to trigger cell death in ALL as a novel treatment strategy. In the first study of this thesis we observed that RSL3, a glutathione (GSH) peroxidase 4 (GPX4) inhibitor, triggered ROS accumulation and lipid peroxidation which contributed to ferroptotic cell death. These observations were based on suppression of RSL3 stimulated cell death using different ferroptosis inhibitors like Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1), as well as iron chelator Deferoxamine (DFO) and the vitamin E derivate α-Tocopherol (α-Toc). RSL3-triggered ROS and lipid peroxide production were also inhibited through Fer-1 and α-Toc. Furthermore, lipoxygenases (LOX) were activated upon RSL3 stimulation and contributed to ferroptotic cell death in ALL as well. Selective inhibition of LOX with the 12/15-LOX inhibitor Baicalein and the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA) abolished RSL3-induced ROS production, lipid peroxidation and cell death. In addition, RSL3 induced lipid peroxide-dependent ferroptotic cell death in FAS-associated Death Domain (FADD)-deficient, death receptor-induced apoptosis resistant cells, demonstrating that ferroptosis might circumvent apoptosis resistance.
The second part of the study revealed that RSL3 and Erastin (Era), a GSH-depleting agent, inhibiting the cystine/glutamate antiporter system xc- and ferroptosis inducer, cooperated with the Smac mimetic BV6 to trigger cell death in ALL cells. RSL3/BV6 and Era/BV6 combination-induced cell death was dependent on ROS accumulation, but independent of caspases and key modulators of necroptosis. RSL3/BV6-treated ALL cells exhibited classical features of ferroptotic cell death with iron-dependency, ROS accumulation and lipid peroxidation which was diminished through either pharmacological inhibition (Fer-1, DFO, α-Toc) or genetic inhibition by overexpressing GPX4. Interestingly, Era/BV6-induced cell death in ALL cells was independent of iron but dependent on ROS accumulation, since α-Toc rescued from Era/BV6-triggered ROS production, lipid peroxidation and cell death. Moreover, inhibition of lipid peroxide formation through the addition of Fer-1 or by overexpressing GPX4 failed to rescue from Era/BV6-triggered cell death, even if Era/BV6-stimulated lipid peroxidation was diminished. Likewise, Fer-1 protected from RSL3/BV6-, but not from Era/BV6-generated ROS production, leading to the assumption that other ROS besides lipid-based ROS contributed to cell death in Era/BV6-treated cells. In summary, while RSL3/BV6 induced ferroptosis in ALL, Era/BV6 stimulated a ROS dependent cell death, which was neither dependent on iron nor caspases or receptor-interacting protein (RIP) kinase 1 nor 3. Additionally, using Erastin alone did not trigger ferroptotic cell death in ALL. Finally, with these two studies we tried to unravel the molecular pathway of ferroptosis by using RSL3 and Erastin as well described ferroptosis stimulators. Here, we demonstrate the possibility of a novel treatment strategy to reactivate programmed cell death by impeding redox homeostasis in ALL.
Since ALL failed to induce ferroptosis upon Erastin treatment, we investigated in the third part of this thesis a new model system to induce ferroptosis upon Erastin and RSL3 exposure. Previous studies revealed that rhabdomyosarcoma (RMS) cells might be susceptible to oxidative stress-induced compounds. To this end, we used Erastin as a prototypic ferroptosis stimulus and GSH-depleting agent and demonstrated that GSH depletion, ROS and lipid ROS accumulation contributed to cell death. Additionally, Fer-1, Lip-1, DFO, lipophilic vitamin E derivate α-Toc and GSH, a cofactor of GPX4, protected from Erastin stimulated ROS accumulation, lipid peroxidation and cell death. Also, the use of a broad spectrum protein kinase C (PKC) inhibitor Bisindolylmaleimide I (Bim1), a PKCα and ß selective inhibitor Gö6976 and siRNA-mediated knockdown of PKCα suppressed Erastin-mediated cell death in RMS. Moreover broad spectrum nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (NOX) inhibitor Diphenyleneiodonium (DPI) and a more selective NOX1/4 isoform inhibitor GKT137831 abrogated Erastin-generated ROS formation, lipid peroxidation and cell death. With this, we demonstrate that RMS are vulnerable to ferroptotic cell death and investigated the molecular mechanism of ferroptosis by unravelling that PKC and NOX could have a pivotal role in ROS-mediated ferroptosis signalling in RMS. In this regard, ferroptosis inducers may act as a possible novel treatment strategy for RMS, especially those with poor clinical outcome.
Resistance in glucocorticoid-induced apoptosis is associated with poor prognosis for long term survival in childhood acute lymphoblastic leukemia (ALL). As Smac mimetics have been shown to reactivate apoptosis by antagonizing Inhibitor of Apoptosis (IAP) proteins, we investigate the potential of the Smac mimetic BV6 to overcome glucocorticoid-resistance in ALL. This study shows that BV6 synergistically cooperates with glucocorticoids to trigger apoptosis and to suppress clonogenic growth of pediatric ALL cells. Of note, the BV6/glucocorticoid combination treatment also induces cell death in cells having defects in the apoptotic signaling cascade by inducing a switch from apoptotic to necroptotic cell death. The clinical relevance of our novel combination treatment is underscored by parallel experiments in primary pediatric ALL samples, in which glucocorticoids and BV6 act together to induce cell death in a synergistic manner. Importantly, the addition of BV6 enhances the anti-leukemic effects of glucocorticoids in an in vivo mouse model of pediatric ALL without causing substantial side effects, highlighting the potency of a BV6/glucocorticoid combination treatment. In contrast, BV6 does not increase cytotoxicity of glucocorticoids against several non-malignant cell types of the lympho-hematopoietic system. Furthermore, we have identified the novel underlying mechanism of BV6/glucocorticoid-induced apoptosis by showing that BV6 and glucocorticoids synergistically act together to promote assembly of the ripoptosome, a RIP1/FADD/caspase-8-containing cell death complex. Ripoptosome assembly is critically required for BV6/Dexamethasone-induced cell death, since genetic silencing of its members, i.e. RIP1, reduces ROS production, caspase activation and most importantly cell death induction. BV6/glucocorticoid combination treatment promotes ripoptosome assembly by inhibition of both of its negative regulators, IAP proteins and cFLIP. Thus, we identify that BV6 and glucocorticoids cooperate together to reduce cIAP1, cIAP2 and XIAP protein levels and cFLIP expression. Ripoptosome formation occurs independently of autocrine/paracrine loops of death receptor ligands, since blocking antibodies for TNFα, TRAIL or CD95L or genetic silencing of their corresponding receptors fail to rescue BV6/glucocorticoid-induced cell death. In summary, this study shows that the Smac mimetic BV6 sensitizes for glucocorticoid-induced apoptosis by promoting ripoptosome assembly with important implications for the treatment of childhood ALL.
Cancer cells, in general and especially Rhabdomyosarcoma (RMS) cells have been reported to be highly susceptible to oxidative stress. Based on this knowledge we examined whether the inhibition of the two main antioxidant defense pathways, i.e. the thioredoxin (TRX) and the glutathione (GSH) system, represents a possible new strategy to induce cell death in RMS. To do so, we combined the -glutamylcysteine synthetase (γGCL) inhibitor buthionine sulfoximine (BSO) or the cystine/glutamate antiporter (xc-) inhibitor erastin (ERA), both GSH depleting enzymes, with the thioredoxinreductase (TrxR) inhibitor auranofin (AUR) to evaluate synergistic cell death in the alveolar RMS (ARMS) cell line RH30 and the embryonal RMS (ERMS) cells RD.
Furthermore, we tried to unravel the underlying molecular mechanisms of AUR/BSO or AUR/ERA treatment in RMS cells. Thereby we showed that AUR/BSO as well as AUR/ERA treatment leads to proteasome inhibition characterized by the accumulation of ubiquitinated proteins, which is in agreement with the already published ability of AUR to inhibit proteasomeassociated deubiquitinases (DUBs) aside from TrxR. As a consequence, the protein levels of ubiquitinated short-lived proteins, like NOXA and MCL-1, increase upon treatment with AUR/BSO or AUR/ERA. Consistently, we could detect an increased binding of NOXA to MCL-1. Interestingly, not only NOXA protein levels but also mRNA levels rise upon treatment, pointing to a transcriptional regulation of pro-apoptotic NOXA through AUR/BSO or AUR/ERA combination treatment. The fact that siRNA mediated knockdown of NOXA rescues cells from combination treatment-induced cell death strengthens the role of NOXA as an important regulator of cell death induction. Apart from proteasome inhibition and subsequent NOXA accumulation, AUR cooperates with BSO or ERA to trigger BAX/BAK activation, which is needed for cell death induction, too. Additionally, loss of mitochondrial membrane potential (MMP) as well as caspase activation and PARP cleavage is detected after treatment of RMS cells with AUR/BSO or AUR/ERA.
Except of apoptotic cell death we also detected features of iron-dependent ferroptosis after treatment with AUR/BSO or AUR/ERA. This is not surprising, since BSO and ERA already have been described to induce ferroptotic cell death. Although lipid peroxidation takes place in both cell lines, only in RH30 cells, cell death seems to be partially ferroptosis-dependent, since especially in this cell line AUR/BSO- or AUR/ERA-induced cell death can be rescued with different ferroptosis inhibitors.
Although both combination treatments, AUR/BSO as well as AUR/ERA, induce production of reactive oxygen species (ROS), only the thiol-containing ROS scavengers GSH and its precursor N-acetylcysteine (NAC), but not the non-thiolcontaining antioxidant α-Tocopherol (α-Toc), consistently prevent proteasome inhibition, NOXA accumulation and cell death.
Additionally, we demonstrated that BSO and ERA abolish AUR-mediated upregulation of GSH thereby releasing the AUR cytotoxic effect on RMS cells, in line with the described ability of cysteines to inhibit the function of AUR. Together, this points to the conclusion that GSH depletion, rather than an increase in ROS levels, is important for AUR/BSO- or AUR/ERA-induced cell death.
In conclusion, through revealing that the antitumor activity of AUR is enhanced in combination with GSH depleting agents, we identified redox homeostasis as a new and promising target for the treatment of RMS cells.
