Identification of a novel synergistic induction of cell death by Smac mimetic and HDAC inhibitors in acute myeloid leukemia cells

Accepted Manuscript Title: Identification of a novel synergistic induction of cell death by smac mimetic and HDAC inhibitors in acute myeloid leukemia cells Author: Sofie Steinwascher, Anne-Lucie Nugues, Hannah Schoeneberger, Simone Fulda PII: DOI: Reference:

S0304-3835(15)00370-5 http://dx.doi.org/doi:10.1016/j.canlet.2015.05.020 CAN 12419

To appear in:

Cancer Letters

Received date: Revised date: Accepted date:

20-4-2015 17-5-2015 22-5-2015

Please cite this article as: Sofie Steinwascher, Anne-Lucie Nugues, Hannah Schoeneberger, Simone Fulda, Identification of a novel synergistic induction of cell death by smac mimetic and HDAC inhibitors in acute myeloid leukemia cells, Cancer Letters (2015), http://dx.doi.org/doi:10.1016/j.canlet.2015.05.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Identification of a novel synergistic induction of cell death by Smac mimetic and HDAC inhibitors in acute myeloid leukemia cells

Sofie Steinwascher1, Anne-Lucie Nugues1, Hannah Schoeneberger1, Simone Fulda1-3

1

Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstr. 3a, 60528 Frankfurt, Germany 2 German Cancer Consortium (DKTK), Heidelberg, Germany 3 German Cancer Research Center (DKFZ), Heidelberg, Germany

Running title: Smac mimetic sensitizes for HDAC inhibitors

To whom correspondence and reprint requests should be addressed: Prof. Dr. Simone Fulda Institute for Experimental Cancer Research in Pediatrics Goethe-University Frankfurt Komturstr. 3a 60528 Frankfurt Tel.: +49 69 67866557 Fax: +49 69 6786659157 Email: [email protected]

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2 Highlights:  Synergistic interaction of Smac mimetic and HDAC inhibitors in AML cell lines 

No synergistic toxicity by combination treatment against normal peripheral blood lymphocytes



Smac mimetic and HDAC inhibitors can trigger necroptosis when caspase activation is blocked

Abstract Inhibitor of Apoptosis (IAP) proteins are expressed at high levels in acute myeloid leukemia (AML) and contribute to resistance to programmed cell death. Here, we report that inhibition of IAP proteins by the small-molecule Smac mimetic BV6 acts together with histone deacetylase (HDAC) inhibitors (HDACIs) such as MS275 or SAHA to trigger cell death in AML cell lines in a synergistic manner, as underscored by calculation of combination index (CI). Also, BV6 and HDACIs cooperate to trigger DNA fragmentation, a marker of apoptotic cell death, and to suppress long-term clonogenic survival of AML cells. In contrast, equimolar concentrations of BV6 and MS275 or SAHA do not synergize to elicit cell death in normal peripheral blood lymphocytes (PBLs), emphasizing some tumor cell selectivity of this combination treatment. Addition of the tumor necrosis factor (TNF)-blocking antibody Enbrel significantly reduces BV6/MS275-induced cell death in the majority of AML cell lines, indicating that autocrine/paracrine TNF signaling contributes to cell death. Remarkably,

the

broad-range

caspase

inhibitor

N-benzyloxycarbonyl-Val-Ala-Asp-

fluoromethylketone (zVAD.fmk) fails to rescue MV4-11, Molm13 and OCI-AML3 cells and even enhances BV6/MS275-mediated cell death, whereas zVAD.fmk reduces BV6/MS275induced cell death in NB4 cells. Annexin-V/propidium iodide (PI) double staining reveals that BV6/MS275 cotreatment predominately increases the percentage of double-positive cells. Of note, the Receptor-Interacting Protein (RIP)1 inhibitor necrostatin-1 (Nec-1) or the Mixed Lineage Kinase Domain-Like protein (MLKL) inhibitor necrosulfonamide (NSA)

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3 significantly reduce BV6/MS275-induced cell death in the presence of zVAD.fmk, suggesting that BV6/MS275 cotreatment triggers necroptosis when caspases are inhibited. Thus, BV6 acts in concert with HDACIs to induce cell death in AML cells and can bypass apoptosis resistance, at least in several AML cell lines, by engaging necroptosis as an alternative route of regulated cell death. The identification of a novel synergism of BV6 and HDACIs has important implications for the development of new treatment strategies for AML.

Keywords: apoptosis; Smac; leukemia; necroptosis

Abbreviations: AML, acute myeloid leukemia; CI, combination index; cIAP, cellular inhibitor of apoptosis protein; FACS, fluorescence-activated cell-sorting analysis; FCS, fetal calf serum; FLT3, Fms-like tyrosine kinase 3; FSC/SSC, forward/side scatter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HDAC, histone deacetylase; HDACIs, HDAC inhibitors; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IAP, Inhibitor of Apoptosis Protein; MLKL, Mixed Lineage Kinase Domain-Like protein; Nec-1, Necrostatin1; NF-B, Nuclear Factor kappaB; NSA, necrosulfonamide; PI, propidium iodide; PBL, peripheral blood lymphocyte; RING, Really Interesting New Gene; RIP1, ReceptorInteracting Protein 1; Smac, second mitochondrial-derived activator of caspases; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1; TRAIL, TNF-related apoptosisinducing ligand; XIAP, X-linked inhibitor of apoptosis; zVAD.fmk, N-benzyloxycarbonylVal-Ala-Asp-fluoromethylketone

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4 1. Introduction AML represents the most frequent form of malignant myeloid neoplasm in adults [1]. Since the efficacy of current therapies is limited especially in resistant cases of AML, novel therapeutic concepts are necessary to improve treatment outcome [1]. Treatment resistance is frequently caused by defects in cell death programs, as most current therapies exert their antileukemic effects by triggering cell death pathways in AML cells [2]. Several distinct forms of programmed cell death are currently known [3]. Apoptosis represents one of the best characterized modes of cell death and in most circumstances depends on the activation of caspases as effector molecules [4]. More recently, necroptosis has been identified as an additional form of regulated cell death that is often engaged upon inhibition of caspases [5]. Critical components of necroptotic signaling comprise RIP1, RIP3 and MLKL [5]. There also exist crosstalks between apoptotic and necroptotic pathways. For example, caspase-8 has been described to interfere with necroptosis by cleaving RIP1 [6]. IAP proteins are a family of antiapoptotic proteins that block programmed cell death via various mechanisms [7]. X-linked inhibitor of apoptosis (XIAP) is known to inhibit caspase activation, while cellular inhibitor of apoptosis protein (cIAP)1 and cIAP2 are involved in regulating Nuclear Factor kappaB (NF-B) signaling via their E3 ubiquitin ligase activity [7]. IAP proteins are neutralized by Second mitochondria-derived activator of caspases (Smac) that is released from the mitochondrial intermembrane space into the cytosol during apoptosis [7]. Since overexpression of IAP proteins has been recorded in AML and correlated with poor prognosis [8-12], IAP proteins are considered as promising targets for therapeutic purposes. To antagonize IAP proteins, small-molecule Smac mimetics that mimick the endogenous Smac protein have been developed in recent years [7]. Smac mimetics neutralize the XIAPimposed inhibition of caspases and also stimulate autoubiquitination and subsequent

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5 proteasomal degradation of IAP proteins harboring a Really Interesting New Gene (RING) domain with E3 ligase activity such as cIAP1, cIAP2 and XIAP [13-15]. Previously, we demonstrated that Smac mimetics sensitize AML cells for cytarabine or demethylating agents [16,17]. Besides demethylating agents, also other epigenetic drugs including HDACIs are currently being explored for the treatment of AML [18]. Searching for new avenues to activate cell death pathways in AML, in the present study we investigated the effect of the small-molecule Smac mimetic BV6, which neutralizes XIAP, cIAP1 and cIAP2 [13], in combination with HDACIs.

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6 2. Materials and Methods Cell culture and chemicals AML cell lines were obtained from ATCC (CEM, Manassas, VA, USA) or 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 mM glutamine (Invitrogen, Karlsruhe, Germany), 1% penicillin/streptomycin (Invitrogen) and 25 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Biochrom). PBLs were isolated from buffy coats of healthy donors by ficoll separation (Biochrom) and cultured in X-VIVO Medium

(Lonza,

Walkersville,

MD)

supplemented

with

10%

FCS

and

1%

penicillin/streptomycin. The study has been approved by the local Ethical Committee. Smac mimetic BV6, which neutralizes XIAP, cIAP1 and cIAP2 [13], was kindly provided by Genentech, Inc. (South San Francisco, CA, USA). Caspase inhibitor zVAD.fmk was obtained from Bachem (Heidelberg, Germany), TNF and Nec-1 from Biomol (Hamburg, Germany), NSA from Toronto Research Chemicals Inc. (North York, CA), MS275 and SAHA from Selleck Chemicals (Houston, TX, USA). Enbrel was kindly provided by Pfizer. All chemicals were purchased by Sigma (Steinheim, Germany) unless indicated otherwise.

Western blot analysis Western blot analysis was performed as described previously [19] using the following antibodies: XIAP (BD Biosciences, Heidelberg, Germany), cIAP1 (R&D Systems, Inc., Wiesbaden, Germany), acetylated histone H3 (Upstate Biotechnology, Lake Placid, NY), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Absave, Harrogate, UK) as well as secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA). Enhanced chemiluminescence was used for detection (Amersham Bioscience,

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7 Freiburg, Germany). All Western blots shown are representative of at least two independent experiments.

Determination of cell death and colony formation Apoptosis was determined by flow cytometric analysis (FACSCanto II, BD Biosciences) of DNA fragmentation of PI-stained nuclei as described previously [19]. Cell death was assessed by forward/side (FSC/SSC) scatter analysis and flow cytometry as described previously [20] or by Annexin-V/PI staining (Roche) according to manufacturer's instructions. For determination of colony formation, cells were treated with BV6 and/or MS275 for 12 hours and then seeded in semisolid culture media (H4100, StemCell Technologies, Vancouver, Canada) as described previously [21]. Colonies were counted after 8-10 days.

Gene silencing Gene silencing by small interfering RNA (siRNA) was performed using Neon Transfection System (Invitrogen, Karlsruhe, Germany) as previously described [22] using Silencer®Select siRNAs against RIP1 (#1: s16651, #2: s16653), RIP3 (#1: s21740, #2: s21741) or MLKL (#1: s47087, #2: s47088).

Statistical analysis Statistical significance was assessed by Student's t-test (two-tailed distribution, two-sample, unequal variance). Drug interactions were analyzed by CI method according to Chou [23] using CalcuSyn software (Biosoft, Cambridge, UK). CI <0.9: synergism, 0.9-1.1: additivity, >1.1: antagonism.

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8 3. Results BV6 and HDACIs synergize to induce cell death in AML cells HDACIs are currently under clinical evaluation in AML [18]. Searching for new Smac mimetic-based combination treatments for the treatment of AML, we tested in several AML cell lines with different molecular characteristics (suppl. Table 1) the effects of HDACIs alone and in combination with the small-molecule Smac mimetic BV6 using low to intermediate drug concentrations that we identified in preliminary experiments (data not shown). Importantly, we found that concomitant administration of BV6 together with MS275 or SAHA synergized to induce cell death compared to treatment with either agent alone (Fig. 1A, 1B, suppl. Tab. 2). Kinetic analysis revealed that BV6 and MS275 or SAHA cooperated to induce cell death in a time-dependent manner (Figure 1C, 1D). Western blot analysis confirmed that treatment with MS275 or SAHA caused acetylation of histone H3, which was taken as indicator for HDAC inhibition (Figure 1E). Together, this set of experiments demonstrates that the Smac mimetic BV6 and HDACIs such as MS275 or SAHA synergistically trigger cell death in AML cells.

BV6 and HDACIs cooperate to trigger DNA fragmentation and to suppress clonogenic survival of AML cells To confirm the synergistic interaction of BV6 and HDACIs we analyzed DNA fragmentation as a marker of apoptotic cell death. Similarly, BV6 and MS275 or SAHA acted in concert to cause DNA fragmentation compared to cells treated with either drug alone (Figure 2A, 2B). Besides these short-time assays, we also performed colony assays to explore the effect of the drug combination on long-term survival. Importantly, BV6 and MS275 cooperated to significantly suppress colony formation compared to cells treated with BV6 or MS275 alone (Figure 2C). This set of experiments demonstrates that BV6 and HDACI act together to

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9 trigger DNA fragmentation and to suppress long-term clonogenic survival of AML cells.

BV6/HDACI cotreatment exerts little cytotoxicity against non-malignant PBLs We extended our experiments to normal PBLs freshly isolated from healthy donors that were used as prototypic non-malignant cells to test the selectivity of the BV6/HDACI combination treatment against malignant cells. BV6 and MS275 or SAHA did not cooperate to induce cell death in PBLs at equimolar concentrations of BV6, MS275 or SAHA that synergized to induce cell death in AML cells (Figure 2D, compare Figure 1A and 1B). These findings point to some tumor cell selectivity of the BV6/HDACI combination treatment.

BV6/MS275-induced cell death partly depends on a TNF autocrine/paracrine loop Smac mimetics are known to stimulate autoubiquitination and proteasomal degradation of IAP proteins that harbor a RING domain [13,14,24,25]. Consistently, we confirmed by Western blotting that treatment with BV6 caused downregulation of cIAP1 and XIAP (Fig. 3A). Since Smac mimetics have been described to engage a TNF-driven autocrine/paracrine loop to induce cell death [13,14,24,25], we used the TNF-blocking antibody Enbrel to test the involvement of TNF/tumor necrosis factor receptor (TNFR)1 signaling. Of note, Enbrel significantly reduced BV6/MS275-induced cell death in OCI-AML3, MV4-11 and Molm13 cells, whereas it failed to recue NB4 cells (Figure 3B). This indicates that autocrine/paracrine TNF/TNFR1 signaling contributes to BV6/MS275-mediated cell death in the majority of the tested cell lines.

BV6/MS275 cotreatment triggers caspase-independent cell death in AML cells when caspase activation is blocked. To explore the question whether caspases are required for BV6/MS275-induced cell death, we

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10 used the broad-range caspase inhibitor zVAD.fmk. Remarkably, zVAD.fmk failed to rescue OCI-AML3, MV4-11 and Molm13 cells from BV6/MS275-induced cell death and significantly decreased BV6/MS275-induced cell death only in NB4 cells (Figure 4). Also, we noted that the addition of zVAD.fmk even further enhanced cell death induction in the three AML cell lines that were not protected against cell death by zVAD.fmk, i.e. OCI-AML3, MV4-11 and Molm13 cells (Figure 4). These findings indicate that BV6/MS275 cotreatment triggers caspase-independent cell death in the majority of the tested cell lines when caspase activation is blocked.

BV6/MS275 cotreatment triggers necroptosis upon caspase inhibition in AML cells. To explore the question whether BV6/MS275 cotreatment induces a caspase-independent, non-apoptotic form of cell death upon caspase inhibition, we determined in parallel by dual color flow cytometry phosphatidylserine exposure on the plasma membrane using Annexin-V staining and loss of plasma membrane integrity using PI staining. This analysis revealed that BV6/MS275 cotreatment in the presence of zVAD.fmk predominately increased the percentage of Annexin-V/PI double-positive cells in OCI-AML3 and MV4-11 cells (Figure 5A), underscoring that BV6/MS275 cotreatment also triggers caspase-independent, nonapoptotic cell death. Since a switch from apoptotic to necroptotic cell death has recently been reported upon caspase inhibition [6], we hypothesize that BV6/MS275 cotreatment engages necroptotic cell death in the presence of zVAD.fmk. To test this hypothesis, we used pharmacological inhibitors to block key components of necroptotic signaling. Importantly, addition of the RIP1 inhibitor Nec-1 significantly reduced the percentage of Annexin-V/PI double-positive cells upon treatment with BV6/MS275/zVAD.fmk (Figure 5A). Similarly, Nec-1 significantly rescued BV6/MS275/zVAD.fmk-induced cell death, when cell death was assessed by

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11 FSC/SSC analysis (Figure 5B). In addition, we used NSA, a pharmacological inhibitor of MLKL [26]. Of note, NSA significantly protected OCI-AML3 and MV4-11 cells from BV6/MS275/zVAD.fmk-induced cell death (Figure 5B). In addition, we used a genetic approach to test the involvement of necroptotic cell death. To this end, we silenced by RNA interference key components of necroptosis signaling, i.e. RIP1, RIP3 and MLKL. Two distinct siRNA sequences were used for each target gene and efficient knockdown was confirmed by Western blotting (Figure 6A-C, upper panels). Importantly,

knockdown

of

RIP1,

RIP3

or

MLKL

significantly

reduced

BV6/MS275/zVAD.fmk-induced cell death (Figure 6A-C, lower panels). Together, these findings emphasize that BV6/MS275 cotreatment can induce necroptosis in AML cells when caspase activation is inhibited.

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12 4. Discussion Defects in cell death programs including overexpression of IAP proteins contribute to treatment resistance and poor outcome in AML [27]. Therefore, novel approaches are necessary to reactivate cell death programs in AML. Here, we show that the Smac mimetic BV6 cooperates with HDACIs to trigger cell death and to suppress long-term clonogenic survival of AML cells. This drug combination is highly synergistic as documented by calculation of CI, emphasizing its potency. In contrast to AML cells, no cooperative cytotoxicity of BV6 together with HDACIs was found against non-malignant PBLs derived from healthy donors, thus pointing to some tumor selectivity of this combination treatment. Previously, we reported that the Smac mimetic BV6 acts in concert with demethylating agents such as 5-azacytidine and decitabine to induce cell death in AML cells [17]. Also, the Smac mimetic birinapant in combination with demethylating agents was shown to target AML stem/progenitor cells [28]. Together with our present study, these findings suggest that the combination of Smac mimetics together with epigenetic modifiers may represent a promising approach for the reactivation of cell death programs in AML cells. Since epigenetic drugs can also induce differentiation, in future it will be interesting to explore whether differentiation processes have an impact on the sensitivity of AML cells towards Smac mimetics. Moreover, our study adds another piece of evidence to the concept that therapeutic targeting of IAP proteins can serve to potentiate the antileukemic activity of therapeutics that are currently being used in the treatment of AML including epigenetic modifiers and chemotherapeutics. High expression levels of IAP proteins including XIAP and cIAP2 have been linked to poor prognosis in AML in several independent studies [8-10,12,27]. Furthermore, we demonstrated that Smac mimetic sensitizes AML cell lines and primary AML samples for cytarabineinduced cell death [16]. In addition, Smac mimetic-based combination therapies with increased antileukemic activity have been recorded for other chemotherapeutic agents such as

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13 etoposide and doxorubicin, kinase inhibitors including Fms-like tyrosine kinase 3 (FLT3) and BCR-ABL inhibitors as well as for the death receptor ligand TNF-related apoptosis-inducing ligand (TRAIL) [29-32]. Beyond AML, we previously demonstrated in several cancer entities that Smac mimetics are potent sensitizers to programmed cell death and prime for death receptor-, chemotherapy- or radiation-induced apoptosis [21,22,33-41]. Also, we reported in the past that HDACIs can sensitize cancer cells to cell death induced by death receptor ligands or anticancer drugs [42-44]. Another key finding of our study resides in the demonstration that cotreatment with BV6 and HDACIs exerts antileukemic activity even in a model of apoptosis resistance by engaging necroptosis as an alternative form of programmed cell death. Several pieces of evidence underscore this conclusion: i) Three out of four AML cell lines, which express essential components of necroptosis signaling [16], die in a caspase-independent manner upon BV6/HDACI cotreatment when apoptosis effector pathways are blocked by the broad-range caspase inhibitor zVAD.fmk. Consistently, treatment with BV6/HDACI/zVAD.fmk predominately increases the percentage of Annexin-V/PI double-positive cells emphasizing that cells die in a non-apoptotic fashion. ii) Upon caspase inhibition, BV6/HDACI cotreatment instead elicits necroptosis in AML cells. This switch to necroptotic cell death is confirmed by rescue experiments, showing that pharmacological inhibitors of RIP1 or MLKL or genetic silencing of RIP1, RIP3 or MLKL protect AML cells against BV6/HDACI-induced cell death. This switch from apoptotic to necroptotic cell death in AML cells in which activation of caspases is blocked is in line with recent reports showing that apoptosis can negatively regulate necroptosis, implying that caspase inhibition promotes necroptosis [6]. Our study has several important implications for the development of Smac mimetic-based combination treatments for AML. First, by showing that BV6 and HDACIs act in concert to elicit cell death in AML cells, our study provides a rationale for exploiting this combination in

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14 experimental protocols in the future. The clinical relevance of such a combination composed of Smac mimetics and HDACIs is emphasized by the fact that epigenetic drugs including HDACIs belong to the portfolio of experimental compounds that are currently under evaluation in early clinical trials in AML. Based on our current work, future studies in primary AML samples and in vivo models of AML are warranted. Second, Smac mimetic in combination with HDACIs may pave the avenue to more effective treatment options to overcome evasion of apoptosis in AML by triggering necroptosis as an alternative cell death program. Treatment resistance of AML is often caused by the inability of cells to undergo apoptosis [27], thus underlining the significance of necroptosis induction by Smac mimetic/HDACIs combination therapy in order to bypass evasion of apoptosis. Third, our data showing that synergistic antileukemic activity can be achieved by using suboptimal concentrations of each class of compounds imply that Smac mimetics can serve as sensitizers for HDACIs, allowing a decrease of the dose of HDACIs that is necessary to achieve therapeutic effects. Smac mimetics are currently being evaluated in early clinical trials alone and in combination therapies [7]. For example, the Smac mimetic birinapant is tested in elderly and relapsed AML patients (www.clinicaltrials.gov). Since monotherapy with Smac mimetics may not be sufficient for effective tumor control, it will likely be critical to rationally exploit synergistic drug interactions. Thus, the identified synergistic drug combination of Smac mimetics and HDACIs represents one important step for therapeutic targeting cell death pathways in AML.

5. Acknowledgements. We thank C. Hugenberg for expert secretarial assistance. This work has been partially supported by grants from the BMBF, IUAP VII (to S.F.) and the Deutsche Krebshilfe (to S.S. and S.F.).

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15

6. Conflict of Interest. The authors declare that they do not have any conflict of interest.

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16 7. References [1]

E. Estey, H. Dohner, Acute myeloid leukaemia, Lancet 368 (2006) 1894-1907.

[2]

S. Fulda, K.M. Debatin, Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy, Oncogene 25 (2006) 4798-4811.

[3]

L. Galluzzi, I. Vitale, J.M. Abrams, E.S. Alnemri, E.H. Baehrecke, M.V. Blagosklonny,

et

al.,

Molecular

definitions

of

cell

death

subroutines:

recommendations of the Nomenclature Committee on Cell Death 2012, Cell Death Differ 19 (2012) 107-120. [4]

R.C. Taylor, S.P. Cullen, S.J. Martin, Apoptosis: controlled demolition at the cellular level, Nat Rev Mol Cell Biol 9 (2008) 231-241.

[5]

P. Vandenabeele, L. Galluzzi, T. Vanden Berghe, G. Kroemer, Molecular mechanisms of necroptosis: an ordered cellular explosion, Nat Rev Mol Cell Biol 11 (2010) 700714.

[6]

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.

[7]

S. Fulda, D. Vucic, Targeting IAP proteins for therapeutic intervention in cancer, Nat Rev Drug Discov 11 (2012) 109-124.

[8]

I. Tamm, S.M. Kornblau, H. Segall, S. Krajewski, K. Welsh, S. Kitada, et al., Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias, Clin Cancer Res 6 (2000) 1796-1803.

[9]

I. Tamm, S. Richter, F. Scholz, K. Schmelz, D. Oltersdorf, L. Karawajew, et al., XIAP expression correlates with monocytic differentiation in adult de novo AML: impact on prognosis, Hematol J 5 (2004) 489-495.

Page 16 of 25

17 [10]

L. Bullinger, F.G. Rucker, S. Kurz, J. Du, C. Scholl, S. Sander, et al., Gene-expression profiling identifies distinct subclasses of core binding factor acute myeloid leukemia, Blood 110 (2007) 1291-1300.

[11]

S.C. Luck, A.C. Russ, U. Botzenhardt, P. Paschka, R.F. Schlenk, H. Dohner, et al., Deregulated apoptosis signaling in core-binding factor leukemia differentiates clinically relevant, molecular marker-independent subgroups, Leukemia 25 (2011) 1728-1738.

[12]

C.J. Hess, J. Berkhof, F. Denkers, G.J. Ossenkoppele, J.P. Schouten, J.J. Oudejans, et al., Activated intrinsic apoptosis pathway is a key related prognostic parameter in acute myeloid leukemia, J Clin Oncol 25 (2007) 1209-1215.

[13]

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.

[14]

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.

[15]

L. Wang, F. Du, X. Wang, TNF-alpha induces two distinct caspase-8 activation pathways, Cell 133 (2008) 693-703.

[16]

J. Chromik, C. Safferthal, H. Serve, S. Fulda, Smac mimetic primes apoptosisresistant acute myeloid leukaemia cells for cytarabine-induced cell death by triggering necroptosis, Cancer Lett 344 (2014) 101-109.

[17]

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.

Page 17 of 25

18 [18]

G. Garcia-Manero, Can we improve outcomes in patients with acute myelogenous leukemia? Incorporating HDAC inhibitors into front-line therapy, Best Pract Res Clin Haematol 25 (2012) 427-435.

[19]

S. Fulda, G. Strauss, E. Meyer, K.M. Debatin, Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicininduced apoptosis in leukemic T cells, Blood 95 (2000) 301-308.

[20]

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.

[21]

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.

[22]

B. Schenk, S. Fulda, Reactive oxygen species regulate Smac mimetic/TNFalphainduced necroptotic signaling and cell death, Oncogene (2015) Apr 13 [E-pub ahead of print].

[23]

T.C. Chou, The median-effect principle and the combination index for quantitation of synergism and antagonism, in: T.C. Chou, (Ed.), Synergism and antagonism in chemotherapy, Academic Press, San Diego, USA, 1991, pp. 61-102.

[24]

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.

[25]

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-mimetic-induced apoptosis, Cancer Cell 12 (2007) 445-456.

Page 18 of 25

19 [26]

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.

[27]

S. Fulda, Exploiting inhibitor of apoptosis proteins as therapeutic targets in hematological malignancies, Leukemia 26 (2012) 1155-1165.

[28]

B.Z. Carter, P.Y. Mak, D.H. Mak, Y. Shi, Y. Qiu, J.M. Bogenberger, et al., Synergistic targeting of AML stem/progenitor cells with IAP antagonist birinapant and demethylating agents, J Natl Cancer Inst 106 (2014) djt440.

[29]

E. Weisberg, A.L. Kung, R.D. Wright, D. Moreno, L. Catley, A. Ray, et al., Potentiation of antileukemic therapies by Smac mimetic, LBW242: effects on mutant FLT3-expressing cells, Mol Cancer Ther 6 (2007) 1951-1961.

[30]

E. Weisberg, A. Ray, R. Barrett, E. Nelson, A.L. Christie, D. Porter, et al., Smac mimetics: implications for enhancement of targeted therapies in leukemia, Leukemia 24 (2010) 2100-2109.

[31]

B.Z. Carter, M. Gronda, Z. Wang, K. Welsh, C. Pinilla, M. Andreeff, et al., Smallmolecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of acute myeloid leukemia cells, Blood 105 (2005) 4043-4050.

[32]

F. Servida, D. Lecis, C. Scavullo, C. Drago, P. Seneci, C. Carlo-Stella, et al., Novel second mitochondria-derived activator of caspases (Smac) mimetic compounds sensitize human leukemic cell lines to conventional chemotherapeutic drug-induced and death receptor-mediated apoptosis, Invest New Drugs 29 (2011) 1264-1275.

[33]

L. Wagner, V. Marschall, S. Karl, S. Cristofanon, K. Zobel, K. Deshayes, et al., Smac mimetic sensitizes glioblastoma cells to Temozolomide-induced apoptosis in a RIP1and NF-kappaB-dependent manner, Oncogene 32 (2013) 988-997.

Page 19 of 25

20 [34]

M. Vogler, H. Walczak, D. Stadel, T.L. Haas, F. Genze, M. Jovanovic, et al., Small molecule XIAP inhibitors enhance TRAIL-induced apoptosis and antitumor activity in preclinical models of pancreatic carcinoma, Cancer Res 69 (2009) 2425-2434.

[35]

B.A. Abhari, S. Cristofanon, R. Kappler, D. von Schweinitz, R. Humphreys, S. Fulda, RIP1 is required for IAP inhibitor-mediated sensitization for TRAIL-induced apoptosis via a RIP1/FADD/caspase-8 cell death complex, Oncogene 32 (2013) 32633273.

[36]

R. Berger, C. Jennewein, V. Marschall, S. Karl, S. Cristofanon, L. Wagner, et al., NF{kappa}B Is Required for Smac Mimetic-Mediated Sensitization of Glioblastoma Cells for {gamma}-Irradiation-Induced Apoptosis, Mol Cancer Ther 10 (2011) 18671875.

[37]

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.

[38]

S. Cristofanon, B.A. Abhari, M. Krueger, A. Tchoghandjian, S. Momma, C. Calaminus, et al., Identification of RIP1 as a critical mediator of Smac mimeticmediated sensitization of glioblastoma cells for Drozitumab-induced apoptosis, Cell Death Dis 6 (2015) e1724.

[39]

F. Basit, R. Humphreys, S. Fulda, RIP1 Protein-dependent Assembly of a Cytosolic Cell Death Complex Is Required for Inhibitor of Apoptosis (IAP) Inhibitor-mediated Sensitization to Lexatumumab-induced Apoptosis, J Biol Chem 287 (2012) 3876738777.

Page 20 of 25

21 [40]

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 (2014) Nov 10 [E-pub ahead of print].

[41]

S.H. Vellanki, A. Grabrucker, S. Liebau, C. Proepper, A. Eramo, V. Braun, et al., Small-molecule XIAP inhibitors enhance gamma-irradiation-induced apoptosis in glioblastoma, Neoplasia 11 (2009) 743-752.

[42]

A. Bangert, S. Cristofanon, I. Eckhardt, B.A. Abhari, S. Kolodziej, S. Hacker, et al., Histone deacetylase inhibitors sensitize glioblastoma cells to TRAIL-induced apoptosis by c-myc-mediated downregulation of cFLIP, Oncogene 31 (2012) 46774688.

[43]

S. Hacker, A. Dittrich, A. Mohr, T. Schweitzer, S. Rutkowski, J. Krauss, et al., Histone deacetylase inhibitors cooperate with IFN-gamma to restore caspase-8 expression and overcome TRAIL resistance in cancers with silencing of caspase-8, Oncogene 28 (2009) 3097-3110.

[44]

S. Hacker, S. Karl, I. Mader, S. Cristofanon, T. Schweitzer, J. Krauss, et al., Histone deacetylase inhibitors prime medulloblastoma cells for chemotherapy-induced apoptosis by enhancing p53-dependent Bax activation, Oncogene 30 (2011) 22752281.

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22 Figure legends Fig. 1. BV6 and HDACIs synergize to induce cell death in AML cells. A and B, AML cells were treated for 72 hours with indicated concentrations of BV6 and/or MS275 (A) or SAHA (B). Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three independent experiments performed in triplicate are shown. C and D, AML cells were treated for indicated times with BV6 and/or MS275 (C) or SAHA (D) (OCI-AML3: 3 M BV6, 1 M MS275, 3 M SAHA; MV4-11: 3 M BV6, 0.6 M MS275, 1.75 M SAHA; Molm13: 3 M BV6, 0.75 M MS275, 1.5 M SAHA; NB4: 150 nM BV6, 0.3 M MS275, 1 M SAHA). Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three independent experiments performed in triplicate are shown; *, P < 0.05, **, P < 0.01, ***, P < 0.001. E, AML cells were treated for 12 hours with MS275 or SAHA (OCI-AML3: 1 M MS275, 3 M SAHA; MV4-11: 0.6 M MS275, 1.75 M SAHA; Molm13: 0.75 M MS275, 1.5 M SAHA; NB4: 0.3 M MS275, 1 M SAHA). Protein expression of acetylated histone H3 (Ac-H3) was assessed by Western blotting, GAPDH served as loading control.

Fig. 2. BV6 and HDACIs cooperate to trigger DNA fragmentation and to suppress clonogenic survival of AML cells A and B, AML cells were treated for 72 hours with BV6 and/or MS275 (A) or SAHA (B) (OCI-AML3: 3 M BV6, 1 M MS275, 3 M SAHA; MV4-11: 3 M BV6, 0.6 M MS275, 1.75 M SAHA; Molm13: 3 M BV6, 0.75 M MS275, 1.5 M SAHA; NB4: 150 nM BV6, 0.3 M MS275, 1 M SAHA). Apoptosis was determined by analysis of DNA fragmentation of PI-stained nuclei using flow cytometry. Mean and SD of three independent experiments performed in triplicate are shown; *, P < 0.05, **, P < 0.01, ***, P < 0.001. C, AML cells were treated for 12 hours with BV6 and/or MS275 (OCI-AML3: 3 M BV6,

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23 0.75 M MS275; MV4-11: 3 M BV6, 0.4 M MS275). Colony formation was assessed as described in Materials and Methods. Data are shown as percentage of untreated control with mean and SD of three independent experiments performed in triplicate; **, P < 0.01. D, PBLs were treated for 72 hours with 3 M BV6 and/or 1 M MS275 or 3 M SAHA. Apoptosis was determined by FSC/SSC analysis and flow cytometry. Mean and SD of one experiment in triplicate are shown, each experiment was performed with a PBL preparation of an individual donor (PBL#1-4).

Fig. 3. BV6/MS275-induced cell death partly depends on a TNF autocrine/paracrine loop. A, AML cells were treated for 6 hours with BV6 and/or MS275 (OCI-AML3: 3 M BV6, 1 M MS275; MV4-11: 3 M BV6, 0.6 M MS275; Molm13: 3 M BV6, 0.75 M MS275; NB4: 150 nM BV6, 0.3 M MS275). Protein levels of cIAP1 and XIAP were assessed by Western blotting, GAPDH served as loading control. B, AML cells were treated for 72 hours with BV6 and/or MS275 (OCI-AML3: 3 M BV6, 1 M MS275; MV4-11: 3 M BV6, 0.6 M MS275; Molm13: 3 M BV6, 0.75 M MS275; NB4: 150 nM BV6, 0.3 M MS275) in the presence or absence of 100 g/ml Enbrel. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three independent experiments performed in triplicate are shown. *, P < 0.05; **, P < 0.01; n.s., not significant.

Fig. 4. BV6/MS275 cotreatment triggers caspase-independent cell death in AML cells when caspase activation is blocked. AML cells were treated for 72 hours with BV6 and/or MS275 in the presence or absence of 20 M zVAD.fmk (OCI-AML3: 3 M BV6, 1 M MS275; MV4-11: 3 M BV6, 0.6 M

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24 MS275; Molm13: 3 M BV6, 0.75 M MS275; NB4: 150 nM BV6, 0.3 M MS275). Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three independent experiments performed in triplicate are shown; *, P < 0.05; ***, P < 0.001; n.s., not significant.

Fig. 5. BV6/MS275 cotreatment triggers necroptosis upon caspase inhibition in AML cells. A, AML cells were treated for 12 hours with BV6 and/or MS275 in the presence or absence of 20 M zVAD.fmk or 30 M Nec-1 (OCI-AML3: 3 M BV6, 1 M MS275; MV4-11: 3 M BV6, 0.6 M MS275). Cell death was determined by Annexin-V/PI staining and flow cytometry. The percentage of Annexin-V (A) and/or PI (P)-positive and -negative cells is shown with mean of three independent experiments performed in triplicate, SD were below 12 %; *, P < 0.05; *+, P < 0.01; ***, P < 0.001. B, AML cells were treated with BV6 and/or MS275 in the presence or absence of 20 M zVAD.fmk, 30 M Nec-1 or 1 M NSA (OCI-AML3: 3 M BV6, 1 M MS275; MV4-11: 3 M BV6, 0.6 M MS275) for 12 hours in order to avoid secondary effects of pharmacological inhibitors upon prolonged incubation. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and SD of three independent experiments performed in triplicate are shown; **, P < 0.01; ***, P < 0.001.

Fig. 6. Knockdown of RIP1, RIP3 or MLKL rescues BV6/MS275-induced cell death upon caspase inhibition. AML cells were transiently transfected with siRNA targeting RIP1 (A), RIP3 (B), MLKL (C) or control siRNA. Protein expression of RIP1, RIP3 or MLKL was analyzed by Western blotting (upper panels). Cells were treated for 18 hours with BV6 and MS275 (OCI-AML3: 3

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25 M BV6, 1 M MS275; MV4-11: 3 M BV6, 0.6 M MS275) in the presence 20 M zVAD.fmk. Cell death was determined by forward/side scatter analysis and flow cytometry (lower panels). Mean + SD of three independent experiments performed in triplicate are shown. *, P < 0.05; **, P < 0.01.

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