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Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells
ARTICLE IN PRESS Cancer Letters ■■ (2016) ■■–■■
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Q2 Original Articles
Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells Q1 Steve Gerges a, Katharina Rohde a, Simone Fulda a,b,c,* a
Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, Komturstr. 3a, 60528 Frankfurt, Germany German Cancer Consortium (DKTK), Heidelberg, Germany c German Cancer Research Center (DKFZ), Heidelberg, Germany b
A R T I C L E
I N F O
Article history: Received 12 January 2016 Received in revised form 17 February 2016 Accepted 19 February 2016 Keywords: Smac Leukemia Apoptosis Necroptosis Demethylating agents
A B S T R A C T
Treatment resistance in acute lymphoblastic leukemia (ALL) is often caused by defects in programmed cell death, e.g. by overexpression of Inhibitor of Apoptosis (IAP) proteins. Here, we report that smallmolecule Smac mimetics (i.e. BV6, LCL161, birinapant) that neutralize x-linked IAP (XIAP), cellular IAP (cIAP)1 and cIAP2 cooperate with demethylating agents (i.e. 5-azacytidine (5AC) or 5-aza-2′-deoxycytidine (DAC)) to induce cell death in ALL cells. Molecular studies reveal that induction of cell death is preceded by BV6-mediated depletion of cIAP1 protein and involves tumor necrosis factor (TNF)α autocrine/ paracrine signaling, since the TNFα-blocking antibody Enbrel significantly reduces BV6/5AC-induced cell death. While BV6/5AC cotreatment induces caspase-3 activation, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) only partly rescues ALL cells from BV6/ 5AC-induced cell death. This indicates that BV6/5AC cotreatment engages non-apoptotic cell death upon caspase inhibition. Indeed, genetic silencing of key components of necroptosis such as ReceptorInteracting Protein (RIP)3 or mixed lineage kinase domain-like (MLKL) in parallel with administration of zVAD.fmk provides a significantly better protection against BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Similarly, concomitant administration of pharmacological inhibitors of necroptosis (i.e. necrostatin-1s, GSK′872, dabrafenib, NSA) together with zVAD.fmk is superior in rescuing cells from BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. These findings demonstrate that in ALL cells BV6/5AC-induced cell death is mediated via both apoptotic and necroptotic pathways. Importantly, BV6/5AC cotreatment triggers necroptosis in ALL cells that are resistant to apoptosis due to caspase inhibition. This opens new perspectives to overcome apoptosis resistance with important implications for the development of new treatment strategies for ALL. © 2016 Published by Elsevier Ireland Ltd.
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Introduction While the overall cure rate for children with ALL, the most common pediatric malignancy, nowadays is relatively high, pa-
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Abbreviations: 5AC, 5-azacytidine; ALL, acute lymphoblastic leukemia; cIAP, cellular inhibitor of apoptosis protein; DAC, 5-aza-2′-deoxycytidine; DNMT, DNA methyltransferase; FADD, FAS-associated Death Domain protein; FCS, fetal calf serum; FSC/SSC, forward/side scatter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IAP, inhibitor of apoptosis protein; MLKL, mixed lineage kinase domain-like protein; Nec-1, necrostatin1; NSA, necrosulfonamide; PI, propidium iodide; RIP, Receptor-Interacting Protein; Smac, second mitochondria-derived activator of caspases; TNF, tumor necrosis factor; TRAIL, TNF-related Apoptosis-Inducing Ligand; XIAP, X-linked Inhibitor of Apoptosis; zVAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. * Corresponding author. Tel.: +49 69 67866557; fax: +49 69 6786659157. E-mail address: [email protected] (S. Fulda).
tients with very high-risk or relapsed disease still have a poor prognosis [1,2]. Treatment failure is frequently due to defects of cancer cells in cell death pathways, which are critically required to mediate the antileukemic activity of cytotoxic therapies [3,4]. This emphasizes the need to develop novel treatment concepts that reactivate programmed cell death. Several forms of programmed cell death have been described in recent years [5]. For example, apoptosis is typically characterized by activation of caspases, a set of enzymes that function as common death effector molecules [6]. Evasion of apoptosis is often caused by overexpression of antiapoptotic proteins [3]. IAP proteins such as XIAP inhibit apoptosis by blocking caspase activation, while cIAP1 and cIAP2, both known as E3 ubiquitin ligases, support survival via ubiquitination events [7]. Besides apoptosis, necroptosis has more recently been identified as another form of programmed cell death [8]. Key signaling components of necroptosis comprise the serine/ threonine kinases RIP1 and RIP3 and the pseudokinase MLKL [8].
http://dx.doi.org/10.1016/j.canlet.2016.02.040 0304-3835/© 2016 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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There are also crosstalks between apoptotic and necroptotic pathways, for example caspase-8 has been described to block necroptosis by cleaving RIP1 [9]. Aberrantly high expression levels of IAP proteins have been documented in a variety of human malignancies including leukemia [10]. In childhood ALL, XIAP protein levels have been reported to be significantly upregulated in leukemic blasts of children diagnosed with ALL compared to normal bone marrow mononuclear cells [11]. Smallmolecule inhibitors have been designed to neutralize IAP proteins, including mimetics of the endogenous mitochondrial protein second mitochondria-derived activator of caspase (Smac) that is released from the mitochondrial intermembrane space into the cytosol and promotes apoptosis by binding and antagonizing IAP proteins [7]. In addition, Smac mimetics trigger autoubiquitination and proteasomal degradation of IAP proteins such as cIAP1 [7]. Several Smac mimetics are currently under evaluation in early clinical trials including LCL161, a second-generation orally bioavailable Smac mimetic with nanomolar affinity for XIAP, cIAP1 and cIAP2 [12]. Epigenetic processes of DNA promoter methylation and histone modification have been reported to contribute to tumorigenesis and treatment resistance [13]. Epigenetic drugs such as demethylating agents are considered as promising new options for the treatment of cancers including ALL [14,15]. Searching for novel treatment strategies for ALL, in the present study we investigated the effect of smallmolecule Smac mimetics in combination with demethylating agents.
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Materials and methods
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Cell culture and chemicals
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ALL cell lines were obtained from DSMZ (Braunschweig, Germany) and were cultured in RPMI 1640 or Dulbecco’s Modified Eagle Medium (DMEM) medium (Life Technologies, Inc., Eggenstein, Germany), supplemented with 10% FCS (fetal calf serum) (Biochrom, Berlin, Germany), 1% sodium pyruvate and 1% penicillin/streptomycin (Invitrogen, Karlsruhe, Germany) and 25 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) (Biochrom). The Smac mimetic BV6 was kindly provided by Genentech, Inc. (South San Francisco, CA, USA), LCL161 by Novartis (Nürnberg, Germany), and birinapant was obtained from Selleckchem (Houston, TX, USA). Caspase inhibitor zVAD.fmk was obtained from Bachem (Heidelberg, Germany), necrostatin-1s (Nec-1s) from Biomol (Hamburg, Germany), GSK′872 from Merck (Darmstadt, Germany), dabrafenib from GlaxoSmithKline (Munich, Germany), and necrosulfonamide (NSA) from Toronto Research Chemicals Inc. (North York, CA). Enbrel was kindly provided by Pfizer. All chemicals were purchased by Sigma-Aldrich (Taufkirchen, Germany) or Carl Roth (Karlsruhe, Germany) unless indicated otherwise.
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Western blot analysis
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Western blot analysis was performed as described previously [16] using the following antibodies: cIAP1 (R&D Systems, Inc., Wiesbaden, Germany), RIP3 (Novus Biologicals, Littleton, CO, USA), MLKL (GeneTex, Irvine, CA, USA), and β-Actin (SigmaAldrich), and secondary antibodies labeled with IRDye infrared dyes were used for fluorescence detection (Odyssey Imaging System, LI-COR Bioscience, Bad Homburg, Germany). All Western blots shown are representative of at least two independent experiments.
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Determination of cell death and caspase-3/7 activity
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Cell death was assessed by forward/side scatter (FSC/SCC) analysis and flow cytometry (FACSCanto II, BD Biosciences) as described previously [17] or by propidium iodide (PI) staining to determine plasma membrane permeability. Caspase-3/7 activity was detected by Cell Event Caspase-3/7 Green Detection Reagent (Life Technologies, Inc.) according to the manufacturer’s instructions and ImageXpress Micro XLS system (Molecular Devices, Biberach an der Riss, Germany).
Statistical analysis
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Statistical significance was assessed by Student’s t-test (two-tailed distribution, two-sample, unequal variance).
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Results BV6 and demethylating agents cooperate to induce cell death in ALL cells Searching for new drug combinations for the treatment of ALL, we tested the effects of the small-molecule Smac mimetic BV6, which antagonizes XIAP, cIAP1 and cIAP2 [19], in the presence and absence of the DNA methyltransferase (DNMT) inhibitor 5AC or DAC. As cellular models we used both the B-cell precursor ALL cell lines Tanoue, KOPN-8 and Reh and the T-cell ALL cell line Jurkat. We found that BV6 and 5AC cooperated to induce cell death in all tested ALL cell lines compared to either agent alone (Fig. 1A). Similarly, BV6 acted in concert with DAC to trigger cell death in ALL cells (Fig. 1B). Since Smac mimetics have been described to cause autoubiquitination and proteasomal degradation of cIAP proteins [19–22], we confirmed on-target engagement by examining the effect of BV6 on expression levels of cIAP1 protein. BV6 downregulated cIAP1 protein levels in all tested ALL cell lines (Fig. 1C). Kinetic studies revealed that BV6/ 5AC cotreatment induced cell death in a time-dependent fashion (Fig. 1D). Assessment of cell death by another assay using PI staining to analyze plasma membrane permeability confirmed the cooperative interaction of BV6 and 5AC to induce cell death in ALL cells (Supplementary Fig. S1). To exclude the possibility that the results are restricted to the Smac mimetic BV6, we tested additional, structurally distinct small-molecule Smac mimetics. Similarly, LCL161 and birinapant cooperated with 5AC or DAC to induce cell death in ALL cells (Fig. 1E,F). Together, this set of experiments demonstrates that Smac mimetics (i.e. BV6, LCL161 and birinapant) and demethylating agents (i.e. 5AC and DAC) cooperatively trigger cell death in ALL cells. BV6/5AC-induced cell death partly depends on caspase activity and TNFα autocrine/paracrine loop To gain insights into the molecular mechanisms of BV6/5AC cotreatment in ALL cells, we focused our subsequent mechanistic studies on the ALL cell line Tanoue. To investigate whether cells die via apoptosis, we determined the activation of caspases as a characteristic feature of apoptotic cell death by analyzing the production of active caspase-3 fragments. Indeed, cotreatment with BV6 and 5AC significantly increased caspase-3 activation (Fig. 2A). To explore whether caspase activation is required for cell death we used the broad-range caspase inhibitor zVAD.fmk. We noted that zVAD.fmk partly reduced BV6/5AC-induced cell death, although zVAD.fmk completely abolished BV6/5AC-triggered caspase-3 activation (Fig. 2B). To investigate whether a TNFα-driven autocrine/paracrine loop is involved in mediating BV6/5AC-induced cell death, we used the TNFα-blocking antibody Enbrel. Enbrel significantly decreased cell death upon cotreatment with BV6/5AC (Fig. 2C). Concomitant administration of Enbrel and zVAD.fmk provided a significantly better protection against BV6/5AC-induced cell death compared to either inhibitor alone (Fig. 2C). This set of experiments indicates that BV6/ 5AC-induced cell death partly depends on caspase activity and TNFα autocrine/paracrine signaling.
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Gene silencing
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Gene silencing by small interfering RNA (siRNA) was performed as previously described [18] using Neon Transfection System (Invitrogen) and Silencer®Select siRNAs against RIP3 (#1: s21740, #2: s21741), MLKL (#1: s47087, #2: s47088) or nontargeting control siRNA (s4390843).
Pharmacological inhibitors of necroptosis rescue ALL cells from BV6/ 5AC-induced cell death upon caspase inhibition Based on our findings demonstrating that BV6/5AC cotreatment can engage caspase-independent pathway(s) to cell death in ALL
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
ARTICLE IN PRESS S. Gerges et al./Cancer Letters ■■ (2016) ■■–■■
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Fig. 1. BV6 and demethylating agents synergize to induce cell death in ALL cells. A and B, ALL cells were treated for 72 hours with indicated concentrations of 5AC (A) or DAC (B) and/or BV6 (Tanoue: 3 μM BV6; KOPN-8: 3 μM BV6; Reh: 0.1 μM BV6; Jurkat: 5 μM BV6). Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of at least three experiments performed in triplicate are shown. (C) ALL cells were treated for three hours with BV6 (Tanoue: 3 μM BV6; KOPN-8: 3 μM BV6; Reh: 0.1 μM BV6; Jurkat: 5 μM BV6). Protein levels of cIAP1 were assessed by Western blotting, β-Actin was used as loading control. (D) Tanoue and KOPN-8 cells were treated for indicated times with 3 μM BV6 and/or 5AC (Tanoue: 10 μM 5AC; KOPN-8: 1 μM 5AC). Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three experiments performed in triplicate are shown; *P < 0.05; ***P < 0.001 comparing BV6/5AC-treated cells to BV6-treated cells. (E) Tanoue cells were treated for 72 hours with indicated concentrations of LCL161 and/or 10 μM 5AC or 5 μM DAC. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of at least three experiments performed in triplicate are shown. (F) Tanoue cells were treated for 72 hours with indicated concentrations of birinapant and/or 10 μM 5AC or 5 μM DAC. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of four experiments performed in triplicate are shown.
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cells when caspases are inhibited, we hypothesized that in the presence of zVAD.fmk BV6/5AC-treated ALL cells undergo necroptotic cell death, since caspase inhibition has previously been reported to cause a switch from apoptotic to necroptotic cell death [9]. To test this hypothesis, we used a pharmacological approach to block critical signaling components of necroptosis. Importantly, concomitant administration of the RIP1 inhibitor Nec-1s and zVAD.fmk caused a further significant reduction of BV6/5AC-induced cell death compared to cells that were treated with BV6/5AC in the presence of zVAD.fmk, but without Nec-1s, whereas treatment with Nec-1s alone failed to protect ALL cells from cell death (Fig. 3A). Also, parallel administration of the RIP3 inhibitors GSK′872 and dabrafenib and zVAD.fmk provided a better protection compared to zVAD.fmk alone against BV6/5AC-induced cell death (Fig. 3B,C). Furthermore, addition of NSA, a pharmacological inhibitor of MLKL [23], significantly enhanced the protection by zVAD.fmk against BV6/5AC-induced cell death, while treatment with NSA alone did not rescue cell death (Fig. 3D). These inhibitor experiments indicate that BV6/5AC cotreatment triggers caspase-dependent cell death in cells that are able to activate caspases, whereas it engages caspase-independent
non-apoptotic cell death when caspase activation is blocked. This points to a switch from apoptosis to necroptosis upon caspase inhibition. Silencing of RIP3 or MLKL protects ALL cells from BV6/5AC-induced cell death upon caspase inhibition In addition to these pharmacological rescue experiments, we used a genetic approach to block key components of necroptosis signaling. To this end, we knocked down RIP3 by RNA interference using two distinct siRNA sequences. Efficient silencing of RIP3 was confirmed by Western blotting (Fig. 4A). Of note, knockdown of RIP3 significantly reduced BV6/5AC/zVAD.fmk-induced cell death compared to BV6/5AC/zVAD.fmk-treated ALL cells that were transfected with control siRNA (Fig. 4B). By comparison, knockdown of RIP3 provided no significant protection against BV6/5AC-mediated cell death in the absence of zVAD.fmk (Fig. 4B), consistent with our findings showing that the RIP3 inhibitors GSK′872 and dabrafenib did not protect ALL cells against BV6/5AC-induced cell death in the absence of zVAD.fmk (Fig. 3B,C).
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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Besides RIP3, we also silenced MLKL as another key component of necroptosis signaling (Fig. 5A). Similarly, MLKL knockdown significantly decreased BV6/5AC/zVAD.fmk-induced cell death compared to BV6/5AC/zVAD.fmk-treated ALL cells that were transfected with control siRNA (Fig. 5B). By comparison, knockdown of MLKL provided no significant protection against BV6/5AC cotreatment in the absence of zVAD.fmk (Fig. 5B), in line with our data showing that the pharmacological MLKL inhibitor NSA did not rescue ALL cells from BV6/5AC-induced cell death (Fig. 3D). Together, these findings demonstrate that RIP3 and MLKL are required for BV6/5ACinduced cell death upon caspase inhibition.
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Fig. 2. BV6/5AC-induced cell death partly depends on caspase activity and TNFα autocrine/paracrine loop. (A) Tanoue cells were treated for 48 hours with 3 μM BV6 and/or 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk. Caspase-3/7 activity was determined by Cell Event Caspase-3/7 Green Detection Reagent and ImageXpress Micro XLS system. Data are shown as mean and SEM of four independent experiments performed in triplicate; *P < 0.05. B, Tanoue cells were treated for 48 hours with 3 μM BV6 and/or 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three experiments performed in triplicate are shown; ***P < 0.001. C, Tanoue cells were treated for 48 hours with 3 μM BV6 and 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk and/or 25 μg/ml Enbrel. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three experiments performed in triplicate are shown; *P < 0.05; **P < 0.01; ***P < 0.001.
Discussion Since treatment resistance in ALL is often caused by evasion of apoptosis [24], new strategies are required to engage programmed cell death in ALL. Here, we report that Smac mimetics cooperate with demethylating agents to induce cell death in ALL cells via both apoptotic and necroptotic pathways. The following pieces of evidence underscore this conclusion: BV6/5AC cotreatment leads to caspase-3 activation and the broad-range caspase inhibitor zVAD.fmk significantly reduces BV6/5AC-induced cell death. However, this protection by zVAD.fmk, although significant, is only partial, indicating that BV6/5AC cotreatment engages caspase-independent, nonapoptotic cell death upon caspase inhibition. Importantly, genetic silencing of key components of necroptosis such as RIP3 or MLKL in parallel with administration of zVAD.fmk provides a significantly better protection against BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Similarly, concomitant administration of pharmacological inhibitors of necroptosis (i.e. Nec1s, GSK′872, dabrafenib, NSA) together with zVAD.fmk is superior in rescuing cells from BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Our findings demonstrating that BV6/5AC cotreatment triggers necroptosis in ALL cells that are resistant to apoptosis due to caspase inhibition imply that BV6/5AC combination therapy can overcome apoptosis resistance by eliciting necroptosis as another form of programmed cell death. In line with these results, we previously reported that Smac mimetic in combination with demethylating agents engages necroptotic cell death in AML cells when caspase activation is blocked [25]. Our data are consistent with recent evidence showing that caspase-8 activity inhibits necroptosis via cleavage of RIP1, one of the crucial signaling elements of necroptosis [9]. Thus, induction of necroptosis may open new perspectives for the development of effective treatment strategies especially in apoptosis-refractory ALL. Interestingly, our data indicate that RIP3 protein, but not RIP3 kinase activity, is also involved in apoptotic signaling, possibly as a scaffold, as RIP3 knockdown, but not the RIP3 inhibitors GSK′872 and dabrafenib, significantly decreases BV6/5AC-induced cell death in the absence of zVAD.fmk. These results are in line with recent evidence showing that RIP3 protein can indeed function as an apoptosis regulator [26]. Furthermore, we demonstrate that BV6/5AC-induced cell death involves TNFα autocrine/paracrine signaling, since the TNFαblocking antibody Enbrel significantly protects against BV6/5ACinduced cell death. These findings are consistent with our recent data showing that inhibition of TNFα signaling by Enbrel recues AML cells from cell death induced by Smac mimetic and demethylating agents [25]. There is more and more evidence emphasizing that IAP proteins are relevant therapeutic targets in childhood ALL [10]. High expression levels of XIAP protein have been associated with poor prednisone response in childhood T-cell ALL [11]. Also, therapeutic inhibition of IAP proteins by Smac mimetics has been reported to prime ALL cells for cell death in response to various cytotoxic stimuli, including anticancer drugs [27], glucocorticoids [28], death receptor ligands such as TNF-related Apoptosis-Inducing Ligand
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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ARTICLE IN PRESS S. Gerges et al./Cancer Letters ■■ (2016) ■■–■■
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Fig. 3. Pharmacological inhibitors of necroptosis rescue ALL cells from BV6/5AC-induced cell death upon caspase inhibition. Tanoue cells were treated for 48 hours with 3 μM BV6 and 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk and/or 20 μM Nec-1s (A), 5 μM GSK′872 (B), 5 μM dabrafenib (C) or 0.5 μM NSA (D). Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three experiments performed in triplicate are shown; *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.
A (TRAIL) [17] and CD95 ligand [29], as well as glutathione-depleting drugs such as buthionine sulfoximine [30]. In the present study, we demonstrate for the first time that Smac mimetics can sensitize ALL cells for demethylating agents. Demethylating drugs are under evaluation in early clinical trials for the treatment of ALL [15]. Also, several Smac mimetics including LCL161 and birinapant are currently being tested in early clinical trials [31]. This underscores the clinical relevance of a combination regimen with Smac mimetics and demethylating drugs. Beyond ALL, we previously reported a synergistic interaction of Smac mimetics with demethylating agents in acute myeloid leukemia (AML) [25], indicating that this combination strategy has also broader implications for leukemia. Furthermore, we identified a treatment strategy to trigger necroptosis as an alternative mode of programmed cell death in apoptosis-resistant forms of ALL that has the potential to be transferred into clinical application, since both Smac mimetics and demethylating agents can be administered to ALL patients. By comparison, in the past we reported as a proof-of-concept that cotreatment with the Smac mimetic BV6 and TNFα can trigger necroptosis in apoptosis-resistant ALL cells that are deficient in FASassociated Death Domain protein (FADD) or caspase-8 [18,32]. However, BV6/TNFα cotreatment is not a feasible strategy for clinical translation, as systemic administration of TNFα can cause severe toxic side effects. Thus, combination treatment with Smac mimetics and demethylating agents represents an alternative approach for necroptosis induction in ALL. In conclusion, cotreatment with Smac mimetics and demethylating agents represents a promising new
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Fig. 4. Silencing of RIP3 protects ALL cells from BV6/5AC-induced cell death upon caspase inhibition. Tanoue cells were transiently transfected with two distinct siRNAs targeting RIP3 or control siRNA. Protein expression of RIP3 was analyzed by Western blotting (A). Cells were treated for 48 hours with 3 μM BV6 and/or 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk and cell death was determined by FSC/ SSC analysis and flow cytometry (B). Mean + SD of three independent experiments performed in triplicate are shown; *P < 0.05; **P < 0.01.
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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Fig. 5. Silencing of MLKL protects ALL cells from BV6/5AC-induced cell death upon caspase inhibition. Tanoue cells were transiently transfected with two distinct siRNAs targeting MLKL or control siRNA. Protein expression of MLKL was analyzed by Western blotting (A). Cells were treated for 48 hours with 3 μM BV6 and/or 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk and cell death was determined by FSC/ SSC analysis and flow cytometry (B). Mean + SD of three independent experiments performed in triplicate are shown; *P < 0.05; **P < 0.01; n.s., not significant.
389 390 strategy to trigger cell death in ALL, which warrants further 391 exploitation. 392 393 Acknowledgements 394 395 This work has been partially supported by grants from the Q4 Q5 Q6 Deutsche Forschungsgemeinschaft, European Community, BMBF, 396 397Q6 Q7 Wilhelm-Sander Stiftung and IAP VII (to S.F.). 398 399 Conflict of interest 400 401 The authors declare that they do not have any conflict of interest. 402 403 Appendix: Supplementary material 404 405 Supplementary data to this article can be found online at doi:10.1016/j.canlet.2016.02.040. 406 407 References 408 409 [1] C.H. Pui, M.V. Relling, J.R. Downing, Acute lymphoblastic leukemia, N. Engl. J. 410 Med. 350 (2004) 1535–1548. 411 [2] M. Stanulla, M. Schrappe, Treatment of childhood acute lymphoblastic leukemia, 412 Semin. Hematol. 46 (2009) 52–63. 413 [3] S. Fulda, Tumor resistance to apoptosis, Int. J. Cancer 124 (2009) 511–515. 414 [4] S. Fulda, Therapeutic opportunities for counteracting apoptosis resistance in 415 childhood leukaemia, Br. J. Haematol. 145 (2009) 441–454. 416 [5] L. Galluzzi, I. Vitale, J.M. Abrams, E.S. Alnemri, E.H. Baehrecke, M.V. Blagosklonny, 417 et al., Molecular definitions of cell death subroutines: recommendations of the 418 Nomenclature Committee on Cell Death 2012, Cell Death Differ. 19 (2012) 419 107–120. 420
[6] S. Fulda, K.M. Debatin, Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy, Oncogene 25 (2006) 4798–4811. [7] S. Fulda, D. Vucic, Targeting IAP proteins for therapeutic intervention in cancer, Nat. Rev. Drug Discov. 11 (2012) 109–124. [8] T. Vanden Berghe, A. Linkermann, S. Jouan-Lanhouet, H. Walczak, P. Vandenabeele, Regulated necrosis: the expanding network of non-apoptotic cell death pathways, Nat. Rev. Mol. Cell Biol. 15 (2014) 135–147. [9] A. Oberst, C.P. Dillon, R. Weinlich, L.L. McCormick, P. Fitzgerald, C. Pop, et al., Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis, Nature 471 (2011) 363–367. [10] S. Fulda, Inhibitor of Apoptosis (IAP) proteins in hematological malignancies: molecular mechanisms and therapeutic opportunities, Leukemia 28 (2014) 1414–1422. [11] P. Hundsdoerfer, I. Dietrich, K. Schmelz, C. Eckert, G. Henze, XIAP expression is post-transcriptionally upregulated in childhood ALL and is associated with glucocorticoid response in T-cell ALL, Pediatr. Blood Cancer 55 (2010) 260–266. [12] L. Zawel, C. Straub, B. Firestone, J. Sullivan, K. Levine, D. Porter, et al., Therapeutic targeting of inhibitor of apoptosis proteins. In: proceedings of the 101st annual meeting of the American Association for Cancer Research; 2010 Apr 17–21; Washington, DC; Philadelphia, PA: AACR, Cancer Res. 70 (78 Suppl.) (2010) 138. [13] P.A. Jones, S.B. Baylin, The epigenomics of cancer, Cell 128 (2007) 683–692. [14] T. Bhatla, J. Wang, D.J. Morrison, E.A. Raetz, M.J. Burke, P. Brown, et al., Epigenetic reprogramming reverses the relapse-specific gene expression signature and restores chemosensitivity in childhood B-lymphoblastic leukemia, Blood 119 (2012) 5201–5210. [15] M.J. Burke, J.K. Lamba, S. Pounds, X. Cao, Y. Ghodke-Puranik, B.R. Lindgren, et al., A therapeutic trial of decitabine and vorinostat in combination with chemotherapy for relapsed/refractory acute lymphoblastic leukemia, Am. J. Hematol. 89 (2014) 889–895. [16] S. Fulda, G. Strauss, E. Meyer, K.M. Debatin, Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells, Blood 95 (2000) 301–308. [17] M. Fakler, S. Loeder, M. Vogler, K. Schneider, I. Jeremias, K.M. Debatin, et al., Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome Bcl-2-mediated resistance, Blood 113 (2009) 1710–1722. [18] B. Schenk, S. Fulda, Reactive oxygen species regulate Smac mimetic/TNFalphainduced necroptotic signaling and cell death, Oncogene 34 (2015) 5796–5806. [19] E. Varfolomeev, J.W. Blankenship, S.M. Wayson, A.V. Fedorova, N. Kayagaki, P. Garg, et al., IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis, Cell 131 (2007) 669–681. [20] M.J. Bertrand, S. Milutinovic, K.M. Dickson, W.C. Ho, A. Boudreault, J. Durkin, et al., cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination, Mol. Cell 30 (2008) 689–700. [21] J.E. Vince, W.W. Wong, N. Khan, R. Feltham, D. Chau, A.U. Ahmed, et al., IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis, Cell 131 (2007) 682–693. [22] S.L. Petersen, L. Wang, A. Yalcin-Chin, L. Li, M. Peyton, J. Minna, et al., Autocrine TNFalpha signaling renders human cancer cells susceptible to Smac-mimeticinduced apoptosis, Cancer Cell 12 (2007) 445–456. Q3 [23] L. Sun, H. Wang, Z. Wang, S. He, S. Chen, D. Liao, et al., Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase, Cell 148 (2012) 213–227. [24] S. Fulda, Exploiting inhibitor of apoptosis proteins as therapeutic targets in hematological malignancies, Leukemia 26 (2012) 1155–1165. [25] L. Steinhart, K. Belz, S. Fulda, Smac mimetic and demethylating agents synergistically trigger cell death in acute myeloid leukemia cells and overcome apoptosis resistance by inducing necroptosis, Cell Death Dis. 4 (2013) e802. [26] K. Newton, D.L. Dugger, K.E. Wickliffe, N. Kapoor, M.C. de Almagro, D. Vucic, et al., Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis, Science 343 (2014) 1357–1360. [27] S. Loeder, M. Fakler, H. Schoeneberger, S. Cristofanon, J. Leibacher, N. Vanlangenakker, et al., RIP1 is required for IAP inhibitor-mediated sensitization of childhood acute leukemia cells to chemotherapy-induced apoptosis, Leukemia 26 (2012) 1020–1029. [28] K. Belz, H. Schoeneberger, S. Wehner, A. Weigert, H. Bonig, T. Klingebiel, et al., Smac mimetic and glucocorticoids synergize to induce apoptosis in childhood ALL by promoting ripoptosome assembly, Blood 124 (2014) 240–250. [29] S. Loeder, A. Drensek, I. Jeremias, K.M. Debatin, S. Fulda, Small molecule XIAP inhibitors sensitize childhood acute leukemia cells for CD95-induced apoptosis, Int. J. Cancer 126 (2010) 2216–2228. [30] H. Schoeneberger, K. Belz, B. Schenk, S. Fulda, Impairment of antioxidant defense via glutathione depletion sensitizes acute lymphoblastic leukemia cells for Smac mimetic-induced cell death, Oncogene 34 (2015) 4032–4043. [31] S. Fulda, Promises and challenges of Smac mimetics as cancer therapeutics, Clin. Cancer Res. 21 (2015) 5030–5036. [32] B. Laukens, C. Jennewein, B. Schenk, N. Vanlangenakker, A. Schier, S. Cristofanon, et al., Smac mimetic bypasses apoptosis resistance in FADD- or caspase-8deficient cells by priming for tumor necrosis factor alpha-induced necroptosis, Neoplasia 13 (2011) 971–979.
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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Q2 Original Articles
Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells Q1 Steve Gerges a, Katharina Rohde a, Simone Fulda a,b,c,* a
Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, Komturstr. 3a, 60528 Frankfurt, Germany German Cancer Consortium (DKTK), Heidelberg, Germany c German Cancer Research Center (DKFZ), Heidelberg, Germany b
A R T I C L E
I N F O
Article history: Received 12 January 2016 Received in revised form 17 February 2016 Accepted 19 February 2016 Keywords: Smac Leukemia Apoptosis Necroptosis Demethylating agents
A B S T R A C T
Treatment resistance in acute lymphoblastic leukemia (ALL) is often caused by defects in programmed cell death, e.g. by overexpression of Inhibitor of Apoptosis (IAP) proteins. Here, we report that smallmolecule Smac mimetics (i.e. BV6, LCL161, birinapant) that neutralize x-linked IAP (XIAP), cellular IAP (cIAP)1 and cIAP2 cooperate with demethylating agents (i.e. 5-azacytidine (5AC) or 5-aza-2′-deoxycytidine (DAC)) to induce cell death in ALL cells. Molecular studies reveal that induction of cell death is preceded by BV6-mediated depletion of cIAP1 protein and involves tumor necrosis factor (TNF)α autocrine/ paracrine signaling, since the TNFα-blocking antibody Enbrel significantly reduces BV6/5AC-induced cell death. While BV6/5AC cotreatment induces caspase-3 activation, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) only partly rescues ALL cells from BV6/ 5AC-induced cell death. This indicates that BV6/5AC cotreatment engages non-apoptotic cell death upon caspase inhibition. Indeed, genetic silencing of key components of necroptosis such as ReceptorInteracting Protein (RIP)3 or mixed lineage kinase domain-like (MLKL) in parallel with administration of zVAD.fmk provides a significantly better protection against BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Similarly, concomitant administration of pharmacological inhibitors of necroptosis (i.e. necrostatin-1s, GSK′872, dabrafenib, NSA) together with zVAD.fmk is superior in rescuing cells from BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. These findings demonstrate that in ALL cells BV6/5AC-induced cell death is mediated via both apoptotic and necroptotic pathways. Importantly, BV6/5AC cotreatment triggers necroptosis in ALL cells that are resistant to apoptosis due to caspase inhibition. This opens new perspectives to overcome apoptosis resistance with important implications for the development of new treatment strategies for ALL. © 2016 Published by Elsevier Ireland Ltd.
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Introduction While the overall cure rate for children with ALL, the most common pediatric malignancy, nowadays is relatively high, pa-
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Abbreviations: 5AC, 5-azacytidine; ALL, acute lymphoblastic leukemia; cIAP, cellular inhibitor of apoptosis protein; DAC, 5-aza-2′-deoxycytidine; DNMT, DNA methyltransferase; FADD, FAS-associated Death Domain protein; FCS, fetal calf serum; FSC/SSC, forward/side scatter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IAP, inhibitor of apoptosis protein; MLKL, mixed lineage kinase domain-like protein; Nec-1, necrostatin1; NSA, necrosulfonamide; PI, propidium iodide; RIP, Receptor-Interacting Protein; Smac, second mitochondria-derived activator of caspases; TNF, tumor necrosis factor; TRAIL, TNF-related Apoptosis-Inducing Ligand; XIAP, X-linked Inhibitor of Apoptosis; zVAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. * Corresponding author. Tel.: +49 69 67866557; fax: +49 69 6786659157. E-mail address: [email protected] (S. Fulda).
tients with very high-risk or relapsed disease still have a poor prognosis [1,2]. Treatment failure is frequently due to defects of cancer cells in cell death pathways, which are critically required to mediate the antileukemic activity of cytotoxic therapies [3,4]. This emphasizes the need to develop novel treatment concepts that reactivate programmed cell death. Several forms of programmed cell death have been described in recent years [5]. For example, apoptosis is typically characterized by activation of caspases, a set of enzymes that function as common death effector molecules [6]. Evasion of apoptosis is often caused by overexpression of antiapoptotic proteins [3]. IAP proteins such as XIAP inhibit apoptosis by blocking caspase activation, while cIAP1 and cIAP2, both known as E3 ubiquitin ligases, support survival via ubiquitination events [7]. Besides apoptosis, necroptosis has more recently been identified as another form of programmed cell death [8]. Key signaling components of necroptosis comprise the serine/ threonine kinases RIP1 and RIP3 and the pseudokinase MLKL [8].
http://dx.doi.org/10.1016/j.canlet.2016.02.040 0304-3835/© 2016 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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There are also crosstalks between apoptotic and necroptotic pathways, for example caspase-8 has been described to block necroptosis by cleaving RIP1 [9]. Aberrantly high expression levels of IAP proteins have been documented in a variety of human malignancies including leukemia [10]. In childhood ALL, XIAP protein levels have been reported to be significantly upregulated in leukemic blasts of children diagnosed with ALL compared to normal bone marrow mononuclear cells [11]. Smallmolecule inhibitors have been designed to neutralize IAP proteins, including mimetics of the endogenous mitochondrial protein second mitochondria-derived activator of caspase (Smac) that is released from the mitochondrial intermembrane space into the cytosol and promotes apoptosis by binding and antagonizing IAP proteins [7]. In addition, Smac mimetics trigger autoubiquitination and proteasomal degradation of IAP proteins such as cIAP1 [7]. Several Smac mimetics are currently under evaluation in early clinical trials including LCL161, a second-generation orally bioavailable Smac mimetic with nanomolar affinity for XIAP, cIAP1 and cIAP2 [12]. Epigenetic processes of DNA promoter methylation and histone modification have been reported to contribute to tumorigenesis and treatment resistance [13]. Epigenetic drugs such as demethylating agents are considered as promising new options for the treatment of cancers including ALL [14,15]. Searching for novel treatment strategies for ALL, in the present study we investigated the effect of smallmolecule Smac mimetics in combination with demethylating agents.
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Materials and methods
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Cell culture and chemicals
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ALL cell lines were obtained from DSMZ (Braunschweig, Germany) and were cultured in RPMI 1640 or Dulbecco’s Modified Eagle Medium (DMEM) medium (Life Technologies, Inc., Eggenstein, Germany), supplemented with 10% FCS (fetal calf serum) (Biochrom, Berlin, Germany), 1% sodium pyruvate and 1% penicillin/streptomycin (Invitrogen, Karlsruhe, Germany) and 25 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) (Biochrom). The Smac mimetic BV6 was kindly provided by Genentech, Inc. (South San Francisco, CA, USA), LCL161 by Novartis (Nürnberg, Germany), and birinapant was obtained from Selleckchem (Houston, TX, USA). Caspase inhibitor zVAD.fmk was obtained from Bachem (Heidelberg, Germany), necrostatin-1s (Nec-1s) from Biomol (Hamburg, Germany), GSK′872 from Merck (Darmstadt, Germany), dabrafenib from GlaxoSmithKline (Munich, Germany), and necrosulfonamide (NSA) from Toronto Research Chemicals Inc. (North York, CA). Enbrel was kindly provided by Pfizer. All chemicals were purchased by Sigma-Aldrich (Taufkirchen, Germany) or Carl Roth (Karlsruhe, Germany) unless indicated otherwise.
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Western blot analysis
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Western blot analysis was performed as described previously [16] using the following antibodies: cIAP1 (R&D Systems, Inc., Wiesbaden, Germany), RIP3 (Novus Biologicals, Littleton, CO, USA), MLKL (GeneTex, Irvine, CA, USA), and β-Actin (SigmaAldrich), and secondary antibodies labeled with IRDye infrared dyes were used for fluorescence detection (Odyssey Imaging System, LI-COR Bioscience, Bad Homburg, Germany). All Western blots shown are representative of at least two independent experiments.
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Determination of cell death and caspase-3/7 activity
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Cell death was assessed by forward/side scatter (FSC/SCC) analysis and flow cytometry (FACSCanto II, BD Biosciences) as described previously [17] or by propidium iodide (PI) staining to determine plasma membrane permeability. Caspase-3/7 activity was detected by Cell Event Caspase-3/7 Green Detection Reagent (Life Technologies, Inc.) according to the manufacturer’s instructions and ImageXpress Micro XLS system (Molecular Devices, Biberach an der Riss, Germany).
Statistical analysis
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Statistical significance was assessed by Student’s t-test (two-tailed distribution, two-sample, unequal variance).
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Results BV6 and demethylating agents cooperate to induce cell death in ALL cells Searching for new drug combinations for the treatment of ALL, we tested the effects of the small-molecule Smac mimetic BV6, which antagonizes XIAP, cIAP1 and cIAP2 [19], in the presence and absence of the DNA methyltransferase (DNMT) inhibitor 5AC or DAC. As cellular models we used both the B-cell precursor ALL cell lines Tanoue, KOPN-8 and Reh and the T-cell ALL cell line Jurkat. We found that BV6 and 5AC cooperated to induce cell death in all tested ALL cell lines compared to either agent alone (Fig. 1A). Similarly, BV6 acted in concert with DAC to trigger cell death in ALL cells (Fig. 1B). Since Smac mimetics have been described to cause autoubiquitination and proteasomal degradation of cIAP proteins [19–22], we confirmed on-target engagement by examining the effect of BV6 on expression levels of cIAP1 protein. BV6 downregulated cIAP1 protein levels in all tested ALL cell lines (Fig. 1C). Kinetic studies revealed that BV6/ 5AC cotreatment induced cell death in a time-dependent fashion (Fig. 1D). Assessment of cell death by another assay using PI staining to analyze plasma membrane permeability confirmed the cooperative interaction of BV6 and 5AC to induce cell death in ALL cells (Supplementary Fig. S1). To exclude the possibility that the results are restricted to the Smac mimetic BV6, we tested additional, structurally distinct small-molecule Smac mimetics. Similarly, LCL161 and birinapant cooperated with 5AC or DAC to induce cell death in ALL cells (Fig. 1E,F). Together, this set of experiments demonstrates that Smac mimetics (i.e. BV6, LCL161 and birinapant) and demethylating agents (i.e. 5AC and DAC) cooperatively trigger cell death in ALL cells. BV6/5AC-induced cell death partly depends on caspase activity and TNFα autocrine/paracrine loop To gain insights into the molecular mechanisms of BV6/5AC cotreatment in ALL cells, we focused our subsequent mechanistic studies on the ALL cell line Tanoue. To investigate whether cells die via apoptosis, we determined the activation of caspases as a characteristic feature of apoptotic cell death by analyzing the production of active caspase-3 fragments. Indeed, cotreatment with BV6 and 5AC significantly increased caspase-3 activation (Fig. 2A). To explore whether caspase activation is required for cell death we used the broad-range caspase inhibitor zVAD.fmk. We noted that zVAD.fmk partly reduced BV6/5AC-induced cell death, although zVAD.fmk completely abolished BV6/5AC-triggered caspase-3 activation (Fig. 2B). To investigate whether a TNFα-driven autocrine/paracrine loop is involved in mediating BV6/5AC-induced cell death, we used the TNFα-blocking antibody Enbrel. Enbrel significantly decreased cell death upon cotreatment with BV6/5AC (Fig. 2C). Concomitant administration of Enbrel and zVAD.fmk provided a significantly better protection against BV6/5AC-induced cell death compared to either inhibitor alone (Fig. 2C). This set of experiments indicates that BV6/ 5AC-induced cell death partly depends on caspase activity and TNFα autocrine/paracrine signaling.
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Gene silencing
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Gene silencing by small interfering RNA (siRNA) was performed as previously described [18] using Neon Transfection System (Invitrogen) and Silencer®Select siRNAs against RIP3 (#1: s21740, #2: s21741), MLKL (#1: s47087, #2: s47088) or nontargeting control siRNA (s4390843).
Pharmacological inhibitors of necroptosis rescue ALL cells from BV6/ 5AC-induced cell death upon caspase inhibition Based on our findings demonstrating that BV6/5AC cotreatment can engage caspase-independent pathway(s) to cell death in ALL
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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Fig. 1. BV6 and demethylating agents synergize to induce cell death in ALL cells. A and B, ALL cells were treated for 72 hours with indicated concentrations of 5AC (A) or DAC (B) and/or BV6 (Tanoue: 3 μM BV6; KOPN-8: 3 μM BV6; Reh: 0.1 μM BV6; Jurkat: 5 μM BV6). Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of at least three experiments performed in triplicate are shown. (C) ALL cells were treated for three hours with BV6 (Tanoue: 3 μM BV6; KOPN-8: 3 μM BV6; Reh: 0.1 μM BV6; Jurkat: 5 μM BV6). Protein levels of cIAP1 were assessed by Western blotting, β-Actin was used as loading control. (D) Tanoue and KOPN-8 cells were treated for indicated times with 3 μM BV6 and/or 5AC (Tanoue: 10 μM 5AC; KOPN-8: 1 μM 5AC). Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three experiments performed in triplicate are shown; *P < 0.05; ***P < 0.001 comparing BV6/5AC-treated cells to BV6-treated cells. (E) Tanoue cells were treated for 72 hours with indicated concentrations of LCL161 and/or 10 μM 5AC or 5 μM DAC. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of at least three experiments performed in triplicate are shown. (F) Tanoue cells were treated for 72 hours with indicated concentrations of birinapant and/or 10 μM 5AC or 5 μM DAC. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of four experiments performed in triplicate are shown.
223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243
cells when caspases are inhibited, we hypothesized that in the presence of zVAD.fmk BV6/5AC-treated ALL cells undergo necroptotic cell death, since caspase inhibition has previously been reported to cause a switch from apoptotic to necroptotic cell death [9]. To test this hypothesis, we used a pharmacological approach to block critical signaling components of necroptosis. Importantly, concomitant administration of the RIP1 inhibitor Nec-1s and zVAD.fmk caused a further significant reduction of BV6/5AC-induced cell death compared to cells that were treated with BV6/5AC in the presence of zVAD.fmk, but without Nec-1s, whereas treatment with Nec-1s alone failed to protect ALL cells from cell death (Fig. 3A). Also, parallel administration of the RIP3 inhibitors GSK′872 and dabrafenib and zVAD.fmk provided a better protection compared to zVAD.fmk alone against BV6/5AC-induced cell death (Fig. 3B,C). Furthermore, addition of NSA, a pharmacological inhibitor of MLKL [23], significantly enhanced the protection by zVAD.fmk against BV6/5AC-induced cell death, while treatment with NSA alone did not rescue cell death (Fig. 3D). These inhibitor experiments indicate that BV6/5AC cotreatment triggers caspase-dependent cell death in cells that are able to activate caspases, whereas it engages caspase-independent
non-apoptotic cell death when caspase activation is blocked. This points to a switch from apoptosis to necroptosis upon caspase inhibition. Silencing of RIP3 or MLKL protects ALL cells from BV6/5AC-induced cell death upon caspase inhibition In addition to these pharmacological rescue experiments, we used a genetic approach to block key components of necroptosis signaling. To this end, we knocked down RIP3 by RNA interference using two distinct siRNA sequences. Efficient silencing of RIP3 was confirmed by Western blotting (Fig. 4A). Of note, knockdown of RIP3 significantly reduced BV6/5AC/zVAD.fmk-induced cell death compared to BV6/5AC/zVAD.fmk-treated ALL cells that were transfected with control siRNA (Fig. 4B). By comparison, knockdown of RIP3 provided no significant protection against BV6/5AC-mediated cell death in the absence of zVAD.fmk (Fig. 4B), consistent with our findings showing that the RIP3 inhibitors GSK′872 and dabrafenib did not protect ALL cells against BV6/5AC-induced cell death in the absence of zVAD.fmk (Fig. 3B,C).
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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A caspase activity (%)
30
*
Besides RIP3, we also silenced MLKL as another key component of necroptosis signaling (Fig. 5A). Similarly, MLKL knockdown significantly decreased BV6/5AC/zVAD.fmk-induced cell death compared to BV6/5AC/zVAD.fmk-treated ALL cells that were transfected with control siRNA (Fig. 5B). By comparison, knockdown of MLKL provided no significant protection against BV6/5AC cotreatment in the absence of zVAD.fmk (Fig. 5B), in line with our data showing that the pharmacological MLKL inhibitor NSA did not rescue ALL cells from BV6/5AC-induced cell death (Fig. 3D). Together, these findings demonstrate that RIP3 and MLKL are required for BV6/5ACinduced cell death upon caspase inhibition.
*
20
10
0 5AC
-
+
-
+
+
BV6
-
-
+
+
+
zVAD.fmk
-
-
-
-
+
B
60 cell death (%)
50
***
***
40 30 20 10
0 5AC
-
+
-
+
BV6
-
-
+
+
+
zVAD.fmk
-
-
-
-
+
C
60
***
cell death (%)
50
+
*
**
**
40
30 20 10
264 265 266 267 268 269 270 271 272 273 274 275 276
0 5AC
-
+
+
+
+
BV6
-
+
+
+
+
Enbrel
-
-
+
-
+
zVAD.fmk
-
-
-
+
+
Fig. 2. BV6/5AC-induced cell death partly depends on caspase activity and TNFα autocrine/paracrine loop. (A) Tanoue cells were treated for 48 hours with 3 μM BV6 and/or 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk. Caspase-3/7 activity was determined by Cell Event Caspase-3/7 Green Detection Reagent and ImageXpress Micro XLS system. Data are shown as mean and SEM of four independent experiments performed in triplicate; *P < 0.05. B, Tanoue cells were treated for 48 hours with 3 μM BV6 and/or 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three experiments performed in triplicate are shown; ***P < 0.001. C, Tanoue cells were treated for 48 hours with 3 μM BV6 and 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk and/or 25 μg/ml Enbrel. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three experiments performed in triplicate are shown; *P < 0.05; **P < 0.01; ***P < 0.001.
Discussion Since treatment resistance in ALL is often caused by evasion of apoptosis [24], new strategies are required to engage programmed cell death in ALL. Here, we report that Smac mimetics cooperate with demethylating agents to induce cell death in ALL cells via both apoptotic and necroptotic pathways. The following pieces of evidence underscore this conclusion: BV6/5AC cotreatment leads to caspase-3 activation and the broad-range caspase inhibitor zVAD.fmk significantly reduces BV6/5AC-induced cell death. However, this protection by zVAD.fmk, although significant, is only partial, indicating that BV6/5AC cotreatment engages caspase-independent, nonapoptotic cell death upon caspase inhibition. Importantly, genetic silencing of key components of necroptosis such as RIP3 or MLKL in parallel with administration of zVAD.fmk provides a significantly better protection against BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Similarly, concomitant administration of pharmacological inhibitors of necroptosis (i.e. Nec1s, GSK′872, dabrafenib, NSA) together with zVAD.fmk is superior in rescuing cells from BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Our findings demonstrating that BV6/5AC cotreatment triggers necroptosis in ALL cells that are resistant to apoptosis due to caspase inhibition imply that BV6/5AC combination therapy can overcome apoptosis resistance by eliciting necroptosis as another form of programmed cell death. In line with these results, we previously reported that Smac mimetic in combination with demethylating agents engages necroptotic cell death in AML cells when caspase activation is blocked [25]. Our data are consistent with recent evidence showing that caspase-8 activity inhibits necroptosis via cleavage of RIP1, one of the crucial signaling elements of necroptosis [9]. Thus, induction of necroptosis may open new perspectives for the development of effective treatment strategies especially in apoptosis-refractory ALL. Interestingly, our data indicate that RIP3 protein, but not RIP3 kinase activity, is also involved in apoptotic signaling, possibly as a scaffold, as RIP3 knockdown, but not the RIP3 inhibitors GSK′872 and dabrafenib, significantly decreases BV6/5AC-induced cell death in the absence of zVAD.fmk. These results are in line with recent evidence showing that RIP3 protein can indeed function as an apoptosis regulator [26]. Furthermore, we demonstrate that BV6/5AC-induced cell death involves TNFα autocrine/paracrine signaling, since the TNFαblocking antibody Enbrel significantly protects against BV6/5ACinduced cell death. These findings are consistent with our recent data showing that inhibition of TNFα signaling by Enbrel recues AML cells from cell death induced by Smac mimetic and demethylating agents [25]. There is more and more evidence emphasizing that IAP proteins are relevant therapeutic targets in childhood ALL [10]. High expression levels of XIAP protein have been associated with poor prednisone response in childhood T-cell ALL [11]. Also, therapeutic inhibition of IAP proteins by Smac mimetics has been reported to prime ALL cells for cell death in response to various cytotoxic stimuli, including anticancer drugs [27], glucocorticoids [28], death receptor ligands such as TNF-related Apoptosis-Inducing Ligand
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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B n.s.
cell death (%)
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*
**
40
**
30 20 10
30 20 10
0 5AC
-
+
+
+
+
0 5AC
-
+
+
+
BV6
-
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+
+
+
BV6
-
+
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+
+
Nec-1s
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+
GSK'872
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zVAD.fmk
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-
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zVAD.fmk
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-
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C 50
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D
***
40 cell death (%)
cell death (%)
30
20
+
*
50
40
**
30 20 10
10 0 5AC
-
+
+
+
+
0 5AC
-
+
+
+
BV6
-
+
+
+
+
BV6
-
+
+
+
+
Dabrafenib
-
-
+
-
+
NSA
-
-
+
-
+
zVAD.fmk
-
-
-
+
+
zVAD.fmk
-
-
-
+
+
+
Fig. 3. Pharmacological inhibitors of necroptosis rescue ALL cells from BV6/5AC-induced cell death upon caspase inhibition. Tanoue cells were treated for 48 hours with 3 μM BV6 and 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk and/or 20 μM Nec-1s (A), 5 μM GSK′872 (B), 5 μM dabrafenib (C) or 0.5 μM NSA (D). Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three experiments performed in triplicate are shown; *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.
A (TRAIL) [17] and CD95 ligand [29], as well as glutathione-depleting drugs such as buthionine sulfoximine [30]. In the present study, we demonstrate for the first time that Smac mimetics can sensitize ALL cells for demethylating agents. Demethylating drugs are under evaluation in early clinical trials for the treatment of ALL [15]. Also, several Smac mimetics including LCL161 and birinapant are currently being tested in early clinical trials [31]. This underscores the clinical relevance of a combination regimen with Smac mimetics and demethylating drugs. Beyond ALL, we previously reported a synergistic interaction of Smac mimetics with demethylating agents in acute myeloid leukemia (AML) [25], indicating that this combination strategy has also broader implications for leukemia. Furthermore, we identified a treatment strategy to trigger necroptosis as an alternative mode of programmed cell death in apoptosis-resistant forms of ALL that has the potential to be transferred into clinical application, since both Smac mimetics and demethylating agents can be administered to ALL patients. By comparison, in the past we reported as a proof-of-concept that cotreatment with the Smac mimetic BV6 and TNFα can trigger necroptosis in apoptosis-resistant ALL cells that are deficient in FASassociated Death Domain protein (FADD) or caspase-8 [18,32]. However, BV6/TNFα cotreatment is not a feasible strategy for clinical translation, as systemic administration of TNFα can cause severe toxic side effects. Thus, combination treatment with Smac mimetics and demethylating agents represents an alternative approach for necroptosis induction in ALL. In conclusion, cotreatment with Smac mimetics and demethylating agents represents a promising new
RIP3
57 kD
GAPDH
36 kD
B
40
*
siCtrl siRIP3 #1
cell death (%)
347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374
*
50
cell death (%)
50
343 344 345 346
5
30
siRIP3 #2
20
* ** *
10
0 5AC
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+
-
+
BV6
-
-
+
+
+
zVAD.fmk
-
-
-
-
+
+
Fig. 4. Silencing of RIP3 protects ALL cells from BV6/5AC-induced cell death upon caspase inhibition. Tanoue cells were transiently transfected with two distinct siRNAs targeting RIP3 or control siRNA. Protein expression of RIP3 was analyzed by Western blotting (A). Cells were treated for 48 hours with 3 μM BV6 and/or 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk and cell death was determined by FSC/ SSC analysis and flow cytometry (B). Mean + SD of three independent experiments performed in triplicate are shown; *P < 0.05; **P < 0.01.
Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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Fig. 5. Silencing of MLKL protects ALL cells from BV6/5AC-induced cell death upon caspase inhibition. Tanoue cells were transiently transfected with two distinct siRNAs targeting MLKL or control siRNA. Protein expression of MLKL was analyzed by Western blotting (A). Cells were treated for 48 hours with 3 μM BV6 and/or 10 μM 5AC in the presence or absence of 20 μM zVAD.fmk and cell death was determined by FSC/ SSC analysis and flow cytometry (B). Mean + SD of three independent experiments performed in triplicate are shown; *P < 0.05; **P < 0.01; n.s., not significant.
389 390 strategy to trigger cell death in ALL, which warrants further 391 exploitation. 392 393 Acknowledgements 394 395 This work has been partially supported by grants from the Q4 Q5 Q6 Deutsche Forschungsgemeinschaft, European Community, BMBF, 396 397Q6 Q7 Wilhelm-Sander Stiftung and IAP VII (to S.F.). 398 399 Conflict of interest 400 401 The authors declare that they do not have any conflict of interest. 402 403 Appendix: Supplementary material 404 405 Supplementary data to this article can be found online at doi:10.1016/j.canlet.2016.02.040. 406 407 References 408 409 [1] C.H. Pui, M.V. Relling, J.R. Downing, Acute lymphoblastic leukemia, N. Engl. J. 410 Med. 350 (2004) 1535–1548. 411 [2] M. Stanulla, M. Schrappe, Treatment of childhood acute lymphoblastic leukemia, 412 Semin. Hematol. 46 (2009) 52–63. 413 [3] S. Fulda, Tumor resistance to apoptosis, Int. J. Cancer 124 (2009) 511–515. 414 [4] S. Fulda, Therapeutic opportunities for counteracting apoptosis resistance in 415 childhood leukaemia, Br. J. Haematol. 145 (2009) 441–454. 416 [5] L. Galluzzi, I. Vitale, J.M. Abrams, E.S. Alnemri, E.H. Baehrecke, M.V. Blagosklonny, 417 et al., Molecular definitions of cell death subroutines: recommendations of the 418 Nomenclature Committee on Cell Death 2012, Cell Death Differ. 19 (2012) 419 107–120. 420
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Please cite this article in press as: Steve Gerges, Katharina Rohde, Simone Fulda, Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.040
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