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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.
Since Inhibitor of Apoptosis (IAP) proteins are frequently dysregulated in different cancer entities and contribute to apoptosis resistance, pharmacological IAP antagonists are considered to be promising agents for the future development of cancer treatment strategies. IAP antagonists are small-molecule drugs that have been designed to mimic the interaction site of IAP proteins with their endogenous inhibitor Second mitochondrial activator of caspases (SMAC). Thus, they are frequently referred to as SMAC mimetics. Treatment with SMAC mimetics engages an apoptotic program in cancers by affecting different components of the apoptotic machinery. Besides disinhibition of caspases, SMAC mimetics trigger non-canonical nuclear factor-κB (NF-κB) signaling, which induces upregulation of tumor necrosis factor (TNF) α and other NF-κB target genes. In particular, TNFα production has been closely linked to the induction of SMAC mimetic-mediated cell death. The TNFα-dependent para/autocrine loop facilitates the formation of a cytosolic complex consisting of caspase-8, Fas-associated death domain (FADD) and Receptor-interacting protein (RIP) 1, which serves as caspase-8 activation platform and ultimately triggers induction of apoptosis. In the present study, we use the small-molecule bivalent SMAC mimetic BV6 to analyze SMAC-stimulated NF-κB signaling in cancer cell lines of different entities. Interestingly, we identify two novel NF-κB-regulated factors that are both required for SMAC mimetic-induced apoptosis in a context-dependent manner. First, we show that NF-κB-dependent upregulation of death receptor 5 (DR5) can serve as an alternative mechanism of BV6-mediated cell death. We demonstrate that BV6 treatment induces NF-κB-dependent but largely TNFα -independent apoptosis in A172 glioblastoma cells. By using an unbiased whole genome expression analysis approach, we identify DR5 as a critical NF-κB target gene, which substitutes TNFα and is indispensable for BV6-initated cell death in A172 cells. Second, we demonstrate that Interferon regulatory factor (IRF) 1 is required for BV6-induced TNFα production and apoptosis. Our study provides evidence that IRF1 closely cooperates with the NF-κB network in BV6-mediated cell death and additionally alters expression of selective SMAC mimetic-induced target genes. Furthermore, we show that BV6 treatment triggers secretion of a set of proinflammatory cytokines and increases attraction of monocytes to BV6-treated tumor cells in an IRF1-dependent manner. In summary, our work supports the notion that NF-κB-regulated factors are critically required for SMAC mimetic-initiated apoptosis. We show that IRF1 is indispensable for TNFα production and cell death in BV6-sensitive cell lines and that also DR5 can serve as a proapoptotic NF-κB-controlled factor in BV6-induced apoptosis besides TNFα. Furthermore, this study contributes to an improved understanding on non-apoptotic functions of SMAC mimetics, as IRF1 additionally influences expression levels of proinflammatory cytokines and attraction of immune cells. Thus, our work provides novel insights into the regulation of SMAC mimetic-induced signaling events, which is crucial for the translation of SMAC mimetics for use in clinical application.
Small molecule inhibitors sensitize neuroblastoma cells for chemotherapeutic drug-induced apoptosis
(2015)
Neuroblastoma (NB) is one of the most common solid extracranial pediatric tumors, deriving from undifferentiated cells of the peripheral nervous system. It accounts for approximately 10% of all childhood cancers. High stage tumors usually show poor prognosis despite aggressive treatment such as radiotherapy or chemotherapy. Therefore, it is of utmost importance to find novel treatment strategies in order to improve existing chemotherapy protocols. Combination treatment offers advantages, as chemotherapeutic drugs can be applied in low and subtoxic doses, reducing possible side-effects. Here, we report in a two-part study that small molecule inhibitors (SMI), namely BI 2536, a PLK1 inhibitor and BV6, a SMAC mimetic (SM), sensitize neuroblastoma cells for chemotherapeutic drug-induced cell death. By using i) BI 2536 in combination with vinca alkaloids and ii) BV6 in combination with either doxorubicin or vinca alkaloids, we show that cell death is synergistically enhanced compared to monotherapy. Furthermore, combination treatment significantly reduces survival of NB cells in long-term assays, compared to single treatment. We identify that vinca alkaloid/SMI combinations induce mitotic arrest, as shown by phosphorylation of histone H3, which results in the induction of intrinsic apoptosis and inhibition of CDK1 by RO-3306 could abolish these findings. Mechanistically, upon vinca alkaloid/SMI-induced mitotic arrest, anti-apoptotic BCL-2 proteins such as MCL-1, BCL-2 or BCL-XL are degraded or inactivated by phosphorylation, which induces the activation of the proapoptotic BCL-2 family proteins BAX and BAK. The importance of the mitochondrial apoptosis pathway in vinca alkaloid/SMI-induced cell death was further highlighted by the fact that ectopic expression of BCL-2 inhibits vinca alkaloid/SMI-induced DNA fragmentation and BAK- and caspase-activation. In contrast to the vinca alkaloid/SMI cotreatment, DOX/SMI (DOX/BV6)-induced apoptosis only partially involves the mitochondrial pathway. Instead, we clarify that RIP1 is required for DOX/BV6-induced apoptosis, as pharmacological and genetic inhibition of RIP1 rescues from apoptosis induction. Although it has been shown in previous studies that SM-treatment (e.g. BV6) can induce the NF-κB pathway and auto-/paracrine TNFα production through cIAP1/2 depletion, DOX/BV6-induced apoptosis is completely independent of NF-κB activation in our setting, despite fast cIAP1 depletion. This conclusion is based on the fact that inhibition of the NF-κB pathway by exogenously expressed dominant-negative IκBα as well as application of a TNFα blocking antibody does not reduce DOX/BV6-induced cell death. In summary, we unravel two new promising treatment strategies for neuroblastoma patients by using a combination treatment of two different small molecule inhibitors, combined with well-characterized chemotherapeutic agents. Furthermore we give detailed insights into cell death pathways induced by these combination treatments, in which mitochondria and RIP1 have a differential role in chemotherapeutic drug-induced apoptosis.
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.
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.
Necroptosis is a programmed cell death pathway that is implicated in a variety of human diseases. In recent years, increasing knowledge has been gained on the necroptotic signaling cascade. Nevertheless, the role of reactive oxygen species (ROS) in necroptosis is still ambiguous. In this study, we reveal that ROS critically regulate BV6/TNFα-induced necroptotic signaling in FADD-deficient Jurkat cells and in zVAD-treated MV4-11 cells. We show that several ROS scavengers such as butylated hydroxyanisole (BHA), N-acetylcysteine (NAC), α-tocopherol (αToc) and ethyl pyruvate (EP) significantly reduce ROS production and BV6/TNFα–induced cell death. Importantly, ROS are produced prior to cell death induction and promote the assembly of the Receptor-interacting protein kinase (RIP)1/RIP3 necrosome complex via a potential positive feedback loop since on the one hand radical scavengers diminish RIP1/RIP3 necrosome formation and since on the other hand RIP1 or RIP3 silencing attenuates ROS production. Furthermore, the deubiquitinase CYLD contributes to BV6/TNFα-induced ROS generation, necrosome assembly and cell death since CYLD knockdown attenuates all these events. Of note, knockdown of the downstream effector protein mixed lineage kinase domain like (MLKL) only partly reduces BV6/TNFα-triggered ROS production and cell death and does not affect necrosome formation. Contrary to expectations, the MLKL inhibitor Necrosulfonamide (NSA) not only decreases BV6/TNFα-stimulated ROS production and cell death but also attenuates RIP1/RIP3 necrosome assembly pointing to additional and MLKL-independent anti-necroptotic effects of NSA. Interestingly, silencing of the potential necroptotic excecutors mitochondrial proteins phosphoglycerate mutase family member 5 (PGAM5) or Dynamin-related protein 1 (Drp1) does not affect BV6/TNFα-induced cell death. Consistently, mitochondrial perturbations are not implicated in BV6/TNFα-induced cell death since mitochondrial membrane potential and respiration remain stable along with to BV6/TNFα-triggered necroptosis induction. Interference with the mitochondrial potential by depolarizing agents such as FCCP reduces BV6/TNFα-induced necroptosis indicating that proper mitochondrial function or a well-defined redox status is required for necroptotic cell death execution. This study demonstrates that ROS are critically involved in BV6/TNFα-induced necroptosis and thus provides novel insights into the redox regulation of necroptotic signaling.
Rhabdomyosarcoma is the most common paediatric soft-tissue sarcoma, and for tumour recurrence, the prognosis is still unfavourable. The current standard therapy consisting of surgery, radiation and combined chemotherapy does not consider the specific biology of this tumour.
Histone deacetylases (HDACs) and the Lysine-specific demethylase-1 (LSD1) are two epigenetic modifiers which are both part of repressor complexes leading to transcriptional silencing of target genes. Whereas HDACs lead to deacetylation of several lysine-residues within the histone tail, LSD1 is specific for demethylation of H3K4me2 and H3K4me1, as well as in a different context for H3K9me2. Rhabdomyosarcoma is reported to harbour high levels of LSD1, but the functional relevance is yet unclear. HDAC inhibition proved to be effective as single agent treatment, however, the proximity of HDAC1/2 and LSD1 in repressor complexes at the DNA implies a suitable rationale for a combination therapy potentially leading to cooperative effects on target gene transcription. In this study, we aimed to evaluate the potential of a combined LSD1 and HDAC inhibition for cell death induction in rhabdomyosarcoma cell lines. Whereas LSD1 inhibitors failed to induce cell death on their own, the combined inhibition of HDACs and LSD1 resulted in highly synergistic cell death induction. This effect extended to several combinations of LSD1 and HDAC inhibitors as well as to four different rhabdomyosarcoma cell lines, two of embryonal and two of alveolar histology.
With the use of the HDAC inhibitor JNJ-26481585 and the reversible LSD1 inhibitor GSK690, we demonstrated that the cell death induced by the combination matches with the details of intrinsic mitochondrial apoptosis. JNJ-26481585/GSK690-induced cell death is partially caspase-dependent and leads to caspase cleavage, followed by substrate cleavage as shown for PARP, as well as loss of the mitochondrial membrane potential.
Furthermore, JNJ-26481585 and GSK690 acted together to transcriptionally upregulate the proapoptotic proteins NOXA, BIM and BMF, which resulted in respective changes on protein level for both cell lines. However, the antiapoptotic BCL-2 family proteins BCL-2, MCL-1 and BCL-xL displayed only minor changes in protein levels upon treatment with GSK690 and JNJ-26481585, which did not rely on transcriptional activity. Therefore, the increase in proapoptotic proteins induces a shift towards proapoptotic signalling at the mitochondrial membrane. This shift is functionally relevant since knockdown of a proapoptotic protein or overexpression of one of the antiapoptotic proteins BCL-2 and MCL-1, as well as a stabilized mutant MCL-1, can significantly protect from GSK690/JNJ-26481585-induced cell death.
Knockdown of the mitochondrial membrane protein BAK, which is directly guarding the mitochondrial membrane integrity, potently protected from GSK690/JNJ-26481585- induced cell death, directly linking the shift in the BCL-2 family proteins to the observed loss of mitochondrial membrane potential and the further downstream activation of caspases. Furthermore, treatment with JNJ-26481585 and GSK690 resulted in a cell cycle arrest in G2/M phase, indicating additional effects on the tumour cells beside apoptosis induction. Taken together, the combined inhibition of LSD1 and HDACs is a promising strategy for rhabdomyosarcoma treatment.