Induction Of Mitochondrial Outer Membrane Permeabilization Research Articles

Venetoclax and dexamethasone synergize with inotuzumab ozogamicin–induced DNA damage signaling in B-lineage ALL

AbstractAdult patients with relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have a dismal prognosis. To improve pharmacotherapy, we analyzed induction of apoptosis by venetoclax and inotuzumab ozogamicin in terms of cytotoxicity and mode of action. Flow cytometry–based analyses of mitochondrial outer membrane permeabilization (MOMP) and ataxia telangiectasia mutated activation demonstrate rapid induction of MOMP by venetoclax and DNA damage signaling by inotuzumab ozogamicin, respectively. In primary ALL samples and patient-derived xenograft (PDX) models, venetoclax and inotuzumab ozogamicin cooperated and synergized in combination with dexamethasone in vitro in all tested samples of ALL. In murine PDX models, inotuzumab ozogamicin, but not venetoclax, induced complete remission in a dose-dependent manner but constantly failed to achieve relapse-free survival. In contrast, combination therapy with venetoclax, dexamethasone, and inotuzumab ozogamicin induced long-term leukemia-free survival and treatment-free survival in all 3 ALL-PDX models tested. These data demonstrate synergistic and highly efficient pharmacotherapy in preclinical models that qualify for evaluation in clinical trials.

Endolysosomal Targeting of Mitochondria Is Integral to BAX-Mediated Mitochondrial Permeabilization during Apoptosis Signaling.

Mitochondrial outer membrane permeabilization (MOMP) is a core event in apoptosis signaling. However, the underlying mechanism of BAX and BAK pore formation remains incompletely understood. We demonstrate that mitochondria are globally and dynamically targeted by endolysosomes (ELs) during MOMP. In response to pro-apoptotic BH3-only protein signaling and pharmacological MOMP induction, ELs increasingly form transient contacts with mitochondria. Subsequently, ELs rapidly accumulate within the entire mitochondrial compartment. This switch-like accumulation period temporally coincides with mitochondrial BAX clustering and cytochrome c release. Remarkably, interactions of ELs with mitochondria control BAX recruitment and pore formation. Knockdown of Rab5A, Rab5C, or USP15 interferes with EL targeting of mitochondria and functionally uncouples BAX clustering from cytochrome c release, while knockdown of the Rab5 exchange factor Rabex-5 impairs both BAX clustering and cytochrome c release. Together, these data reveal that EL-mitochondrial inter-organelle communication is an integral regulatory component of functional MOMP execution during cellular apoptosis signaling.

Involvement of the biogenic active amine agmatine in mitochondrial membrane permeabilization and release of pro-apoptotic factors.

Agmatine (AGM) produces a dual effect on the mitochondrial permeability transition (MPT) mechanism in rat liver mitochondria: at low concentrations, it induces the phenomenon, at high ones, inhibits it. The prevention at high concentrations is evidenced by the significant inhibition of mitochondrial swelling induced by Ca2+ and phosphate; in this condition, AGM both prevents the release of Apoptosis Inducing Factor (AIF) and enhances the release of other pro-apoptotic factors, such as cytochrome c (cyt c) and Smac/DIABLO. As these factors are released without MPT induction, the involvement of mitochondrial outer membrane permeabilization (MOMP) could be hypothesized. Cyclosporin A (CsA), a powerful inhibitor of MPT, enhanced the AGM-mediated inhibition of swelling, and surprisingly, prevented the release of cyt c and Smac/DIABLO. In the presence of Ca2+, AGM also activated the Bcl-2 family protein Bax, a key factor in inducing MOMP, which is inactivated by CsA. Together with the voltage-dependent anion channel (VDAC), Bax forms channels in the outer membrane further supporting the involvement of MOMP in the release of pro-apoptotic factors. In view of the fact that VDAC was inactivated by ruthenium red, which in turn inhibited the release of cyt c, it can be hypothesized that, on the one hand, AGM inhibits MPT induction and, on the other, it selectively permeabilizes the outer membrane via MOMP induction.

Optimized Induction of Mitochondrial Apoptosis By Combination Therapies with Venetoclax for Chemotherapy-Free Treatment of BCR-ABL+ Acute Lymphoblastic Leukemia in Preclinical Models

Background: BCR-ABL+ acute lymphoblastic leukemia (ALL) in adults has a poor prognosis with allogeneic stem cell transplantation (SCT) considered the best curative option for suitable patients. BH3 mimetics induce mitochondrial outer membrane permeabilization (MOMP) linked to apoptosis induction by releasing BH3-only proteins BIM and/or BID from the anti-apoptotic factors BCL2, BCL-XL, MCL1, BCLW and BFL1. The BCL2-specific BH3 mimetic venetoclax (ABT-199) may provide an opportunity to improve pharmacotherapy of BCR-ABL+ ALL in particular for elderly patients not suitable for SCT.Aim: We aimed to rationally design optimized combination therapies for BCR-ABL+ ALL based on the molecular mechanisms of apoptosis induction by BH3 mimetics.Methods: We first biochemically characterized binding of BIM, a BH3 only activator of mitochondrial apoptosis, to BCL2, BCLXL and MCL1 and its release by BH3 mimetics in two BCR-ABL+ ALL cell lines. We next visualized and quantitated MOMP-induction by BH3 mimetics in viable cells. We then characterized the effects of dexamethasone and tyrosine kinase inhibitors (TKI) imatinib and dasatinib on BIM expression and calculated dose-effect combination indices (CI) for combination therapies in cell lines and two BCR-ABL+ ALL primograft models co-cultured with mesenchymal stem cells ex vivo. We finally used in vivo bioluminescence and survival analyses in murine xenotransplantation models to evaluate therapeutic efficacy in vivo.Results: In BCR-ABL+ BV173 and SUPB15 cells BIM but not BID binds to BCL2. BIM is rapidly released from BCL2 by venetoclax in a time and dose dependent manner. Release of BIM induces both MOMP (as defined by a decrease in mitochondrial membrane potential) and apoptosis (as defined by PARP cleavage and propidium iodide staining). Furthermore, BIM is strongly required for cytotoxicity of venetoclax, dasatinib and dexamethasone. Primary BCR-ABL+ ALL cells are more resistant against MOMP induction by venetoclax than BCR-ABL-negative ones, and BIM expression is reduced in these cells. Both, TKIs and dexamethasone augment BIM expression in BV173 and SUPB15 cells and act synergistically with venetoclax in cell lines and two BCR-ABL+ primografts (CI for the triple combination therapy of venetoclax, dexamethasone and dasatinib between 0.1 and 0.15, CI< 1.0 considered synergistic). Triple combinations with venetoclax, dexamethasone and TKIs efficiently attenuate leukemia progression in xenotransplantation models and, notably, the dasatinib- but not the imatinib-containing combination led to treatment- and leukemia-free long-term survival in a BCR-ABL+ mouse model.Conclusions: These data demonstrate efficacy of venetoclax in ALL. Although BCR-ABL inhibits venetoclax cytotoxicity, this inhibition can be overcome by triple combination therapy with venetoclax, dexamethasone and dasatinib. Since the triple combination therapy can be curative in preclinical xenotranplatation models clinical studies with oral chemotherapy-free regimens may be considered in particular for elderly patients not suitable for allogeneic SCT. DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees.

BAX/BAK-Induced Apoptosis Results in Caspase-8-Dependent IL-1β Maturation in Macrophages

IL-1β is a cytokine of pivotal importance to the orchestration of inflammatory responses. Synthesized as an inactive pro-cytokine, IL-1β requires proteolytic maturation to gain biological activity. Here, we identify intrinsic apoptosis as a non-canonical trigger of IL-1β maturation. Guided by the discovery of the immunomodulatory activity of vioprolides, cyclic peptides isolated from myxobacteria, we observe IL-1β maturation independent of canonical inflammasome pathways, yet dependent on intrinsic apoptosis. Mechanistically, vioprolides inhibit MCL-1 and BCL2, which in turn triggers BAX/BAK-dependent mitochondrial outer membrane permeabilization (MOMP). Induction of MOMP results in the release of pro-apoptotic factors initiating intrinsic apoptosis, as well as the depletion of IAPs (inhibitors of apoptosis proteins). IAP depletion, in turn, operates upstream of ripoptosome complex formation, subsequently resulting in caspase-8-dependent IL-1β maturation. These results establish the ripoptosome/caspase-8 complex as a pro-inflammatory checkpoint that senses the perturbation of mitochondrial integrity.

Dysfunction of Membrane Trafficking Leads to Ischemia-Reperfusion Injury After Transient Cerebral Ischemia

Neurons require an extraordinarily high level of membrane trafficking activities because of enriched axonal terminals and dendritic branches. For that reason, defects in the membrane trafficking pathway are a hallmark of most, and may be all, neurodegenerative disorders. A major cellular membrane trafficking pathway is the Golgi apparatus (Golgi hereafter)-late endosome-lysosome axis for supplying lysosomal enzymes. This pathway is regulated by N-ethylmaleimide-sensitive factor (NSF) ATPase. This review article is to discuss a novel hypothesis that brain ischemia inactivates NSF ATPase, resulting in a cascade of events of disruption of the Golgi-endosome-lysosome pathway, release of cathepsin B (CTSB), and induction of mitochondrial outer membrane permeabilization (MOMP) during the postischemic phase. This hypothesis is supported by recent studies demonstrating that NSF is trapped into inactive protein aggregates in neurons destined to die after brain ischemia. Consequently, Golgi, transport vesicles (TVs), and late endosomes (LEs) are accumulated and damaged, which is followed by CTSB release from these damaged structures. Moderate release of CTSB cleaves Bax-like BH3 protein (Bid) to become active truncated Bid (tBid). Active tBid is then translocated to the mitochondrial outer membrane, resulting in oligomerization of BCL2-associated X protein (Bax) forming the mitochondrial outer membrane pores, and releasing mitochondrial intramembranous proteins. Extensive CTSB release, however, can digest cellular proteins indiscriminately to induce cell death. Based on these new observations, we propose a novel hypothesis, i.e., brain ischemia leads to NSF inactivation, resulting in a massive buildup of damaged Golgi, TVs and LEs, fatal release of CTSB, induction of MOMP, and eventually brain ischemia-reperfusion injury.

Mitochondrial outer membrane permeabilization increases reactive oxygen species production and decreases mean sperm velocity but is not associated with DNA fragmentation in human sperm.

Does induction of mitochondrial outer membrane permeabilization (MOMP) in vitro affect specific functional parameters of human spermatozoa? Our findings show that MOMP induction increases intracellular reactive oxygen species (ROS) and decreases mean sperm velocity but does not alter DNA integrity. MOMP in somatic cells is related to a variety of apoptotic traits, such as alteration of mitochondrial membrane potential (ΔΨm), and increase in ROS production and DNA fragmentation. Although the presence of these apoptotic features has been reported in spermatozoa, to date the effects of MOMP on sperm function and DNA integrity have not been analysed. The study included spermatozoa from fertile donors. Motile sperm were obtained using the swim-up method. The highly motile sperm were collected and diluted with human tubal fluid to a final cell concentration of 5 × 10(6) ml(-1). To induce MOMP, selected sperm were treated at 37°C for 4 h with a mimetic of a Bcl-2 pro-apoptotic protein, ABT-737. MOMP was evaluated by relocating of cytochrome c. In addition, the effect of ABT-737 on mitochondrial inner membrane permeabilization was assessed using the calcein-AM/cobalt chloride method. In turn, ΔΨm was evaluated with JC-1 staining, intracellular ROS production with dihydroethidium, sperm motility was analysed by computer-assisted sperm analysis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Measurements were performed by flow cytometry. MOMP was associated with ΔΨm dissipation (P < 0.05), increased ROS production (P < 0.05) and decreased mean sperm velocity (P < 0.05), but it was not associated with DNA fragmentation. MOMP did not induce a large increase in ROS, which could explain the negligible effect of MOMP on sperm DNA fragmentation under our experimental conditions. The study was carried out in vitro using highly motile sperm, selected by swim-up, from healthy donors. The results obtained in this study reveal that the alterations of sperm functions caused by MOMP are sufficiently relevant to justify its future study in male infertility. None. The study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT (F.T.). The authors declare no conflict of interest.

Novel combination of mitochondrial division inhibitor 1 (mdivi-1) and platinum agents produces synergistic pro-apoptotic effect in drug resistant tumor cells.

Overcoming platinum drug resistance represents a major clinical challenge in cancer treatment. We discovered a novel drug combination using cisplatin and a class of thioquinazolinone derivatives including mdivi-1 (mitochondrial division inhibitor-1), that induces synergistic apoptosis in platinum resistant tumor cells, including those from cisplatin-refractory endstage ovarian cancer patients. However, through study of the combination effect on Drp1 (the reported target of mdivi-1) knockout MEF cells and the functional analysis of mdivi-1 analogs, we revealed that the synergism between mdivi-1 and cisplatin is Drp1-independent. Mdivi-1 impairs DNA replication and its combination with cisplatin induces a synergistic increase of replication stress and DNA damage, causing a preferential upregulation of a BH3-only protein Noxa. Mdivi-1 also represses mitochondrial respiration independent of Drp1, and the combination of mdivi-1 and cisplatin triggers substantial mitochondrial uncoupling and swelling. Upregulation of Noxa and simultaneous mitochondrial swelling causes synergistic induction of mitochondrial outer membrane permeabilization (MOMP), proceeding robust mitochondrial apoptotic signaling independent of Bax/Bak. Thus, the novel mode of MOMP induction by the combination through the "dual-targeting" potential of mdivi-1 on DNA replication and mitochondrial respiration suggests a novel class of compounds for platinum-based combination option in the treatment of platinum as well as multidrug resistant tumors.

TWEAK Induces Apoptosis through a Death-signaling Complex Comprising Receptor-interacting Protein 1 (RIP1), Fas-associated Death Domain (FADD), and Caspase-8

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TNFSF12, CD255) (TWEAK) can stimulate apoptosis in certain cancer cells. Previous studies suggest that TWEAK activates cell death indirectly, by inducing TNFα-mediated autocrine signals. However, the underlying death-signaling mechanism has not been directly defined. Consistent with earlier work, TWEAK assembled a proximal signaling complex containing its cognate receptor FN14, the adaptor TRAF2, and cellular inhibitor of apoptosis protein 1 (cIAP1). Neither the death domain adaptor Fas-associated death domain nor the apoptosis-initiating protease caspase-8 associated with this primary complex. Rather, TWEAK induced TNFα secretion and TNF receptor 1-dependent assembly of a death-signaling complex containing receptor-interacting protein 1 (RIP1), FADD, and caspase-8. Knockdown of RIP1 by siRNA prevented TWEAK-induced association of FADD with caspase-8 but not formation of the FN14-TRAF2-cIAP1 complex and inhibited apoptosis activation. Depletion of the RIP1 E3 ubiquitin ligase cIAP1 enhanced assembly of the RIP1-FADD-caspase-8 complex and augmented cell death. Conversely, knockdown of the RIP1 deubiquitinase CYLD inhibited these functions. Depletion of FADD, caspase-8, BID, or BAX and BAK but not RIP3 attenuated TWEAK-induced cell death. Pharmacologic inhibition of the NF-κB pathway or siRNA knockdown of RelA attenuated TWEAK induction of TNFα and association of RIP1 with FADD and caspase-8. These results suggest that TWEAK triggers apoptosis by promoting assembly of a RIP1-FADD-caspse-8 complex via autocrine TNFα-TNFR1 signaling. The proapoptotic activity of TWEAK is modulated by cIAP1 and CYLD and engages both the extrinsic and intrinsic signaling pathways.

Apoptosis and oncogenesis: give and take in the BCL-2 family

The mitochondrial pathway of apoptosis constitutes one of the main safeguards against tumorigenesis. The BCL-2 family includes the central players of this pathway that regulate cell fate through the control of mitochondrial outer membrane permeabilization (MOMP), and important progress has been made in understanding the dynamic interactions between pro-apoptotic and anti-apoptotic BCL-2 proteins. In particular, recent studies have delineated a stepwise model for the induction of MOMP. BCL-2 proteins are often dysregulated in cancer, leading to increased survival of abnormal cells; however, recent studies have paradoxically shown that apoptosis induction can under some circumstances drive tumor formation, perhaps by inducing compensatory proliferation under conditions of cellular stress. These observations underline the complexity of BCL-2 protein function in oncogenesis.

Cell Stress and Cell Death

Cell Stress and Cell Death

Cleavage of Bid by Executioner Caspases Mediates Feed Forward Amplification of Mitochondrial Outer Membrane Permeabilization during Genotoxic Stress-induced Apoptosis in Jurkat Cells

The extent to which the BH3-only protein Bid is important for intrinsic (mitochondria-mediated) apoptotic cell death induced by genotoxic stress remains controversial. In the present study, we examine this issue using a panel of gene-manipulated Bax-deficient Jurkat T-lymphocytes. Cells stably depleted of Bid were far less sensitive than control-transfected cells to etoposide-induced apoptosis. In particular, drug-induced Bak activation, cytochrome c release, loss of mitochondrial membrane potential, and caspase activation were all decreased in cells lacking Bid. Reconstitution experiments using recombinant proteins and permeabilized Bid-deficient cells demonstrated that truncated Bid (tBid), but not full-length Bid, potently induced Bak activation and the release of cytochrome c. Further, caspase-8-deficient Jurkat cells efficiently cleaved Bid and were sensitive to drug-induced apoptosis. By comparison, Apaf-1-deficient cells, as well as cells overexpressing full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP, failed to cleave Bid in response to genotoxic stress. These data suggest that tBid plays an important regulatory role in the execution of DNA damage-induced cytochrome c release and apoptosis. However, the fact that cleavage of Bid to tBid is mediated by executioner caspases suggests that a self-amplifying feed forward loop involving caspases, Bid, and mitochondria may help determine irreversible commitment to apoptosis.

Endophilin B1/Bif-1 Stimulates BAX Activation Independently from Its Capacity to Produce Large Scale Membrane Morphological Rearrangements

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes. In this reconstituted model system the N-BAR domain of Bif-1 reproduced the stimulatory effect of Bif-1 on functional BAX activation. This process was dependent on physical interaction between Bif-1 N-BAR and BAX as well as on the presence of the mitochondrion-specific lipid cardiolipin. Despite that Bif-1 N-BAR produced large scale morphological rearrangements in MOM-like liposomes, this phenomenon could be separated from functional BAX activation. Furthermore, DLP1 also caused global morphological changes in MOM-like liposomes, but DLP1 did not stimulate BAX-permeabilizing function in the absence or presence of Bif-1. Taken together, our findings not only provide direct evidence for a functional interplay between Bif-1, BAX, and cardiolipin during MOMP but also add significantly to the growing body of evidence indicating that components of the mitochondrial morphogenesis machinery possess proapoptotic functions that are independent from their recognized roles in normal mitochondrial dynamics.

Disruption of the hexokinase–VDAC complex for tumor therapy

Unlike mitochondria from most normal tissues, cancer cell mitochondria demonstrate an association between the glycolytic enzyme hexokinase (HK) and the voltage-dependent anion channel (VDAC). This provides a therapeutic opportunity, as the association appears to protect tumor cells from mitochondrial outer membrane permeabilization (MOMP), an event that marks the point of no return in multiple pathways leading to cell death. In this issue of Oncogene, the plant hormone methyl jasmonate (MJ) is shown to disrupt the interaction between human HK and VDAC, causing the inhibition of glycolysis and the induction of MOMP. MJ has already been shown to have selective anticancer activity in preclinical studies, and this finding may stimulate the development of a novel class of small anticancer compounds that inhibit the HK-VDAC interaction.

Differential contribution of Puma and Noxa in dual regulation of p53-mediated apoptotic pathways

The activation of tumor suppressor p53 induces apoptosis or cell cycle arrest depending on the state and type of cell, but it is not fully understood how these different responses are regulated. Here, we show that Puma and Noxa, the well-known p53-inducible proapoptotic members of the Bcl-2 family, differentially participate in dual pathways of the induction of apoptosis. In normal cells, Puma but not Noxa induces mitochondrial outer membrane permeabilization (MOMP), and this function is mediated in part by a pathway that involves calcium release from the endoplasmic reticulum (ER) and the subsequent caspase activation. However, upon E1A oncoprotein expression, cells also become susceptible to MOMP induction by Noxa, owing to their sensitization to the ER-independent pathway. These findings offer a new insight into differential cellular responses induced by p53, and may have therapeutic implications in cancer.

The pathophysiology of mitochondrial cell death.

In the mitochondrial pathway of apoptosis, caspase activation is closely linked to mitochondrial outer membrane permeabilization (MOMP). Numerous pro-apoptotic signal-transducing molecules and pathological stimuli converge on mitochondria to induce MOMP. The local regulation and execution of MOMP involve proteins from the Bcl-2 family, mitochondrial lipids, proteins that regulate bioenergetic metabolite flux, and putative components of the permeability transition pore. MOMP is lethal because it results in the release of caspase-activating molecules and caspase-independent death effectors, metabolic failure in the mitochondria, or both. Drugs designed to suppress excessive MOMP may avoid pathological cell death, and the therapeutic induction of MOMP may restore apoptosis in cancer cells in which it is disabled. The general rules governing the pathophysiology of MOMP and controversial issues regarding its regulation are discussed.