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The caspase-8 inhibitor emricasan combines with theSMAC mimetic birinapant to induce necroptosis andtreat acute myeloid leukemiaGabriela Brumatti,1,2* Chunyan Ma,1,2* Najoua Lalaoui,1,2 Nhu-Y Nguyen,3 Mario Navarro,4

Maria C. Tanzer,1,2 Jennifer Richmond,5 Margherita Ghisi,6 Jessica M. Salmon,6 Natasha Silke,1,2

Giovanna Pomilio,3 Stefan P. Glaser,1,2 Elisha de Valle,7,8 Raffi Gugasyan,7,8 Mark A. Gurthridge,3

Stephen M. Condon,9 Ricky W. Johnstone,6 Richard Lock,5 Guy Salvesen,4 Andrew Wei,3

David L. Vaux,1,2 Paul G. Ekert,1,2,10†‡ John Silke1,2†‡

Resistance to chemotherapy is a major problem in cancer treatment, and it is frequently associated with failureof tumor cells to undergo apoptosis. Birinapant, a clinical SMAC mimetic, had been designed to mimic the in-teraction between inhibitor of apoptosis proteins (IAPs) and SMAC/Diablo, thereby relieving IAP-mediated cas-pase inhibition and promoting apoptosis of cancer cells. We show that acute myeloid leukemia (AML) cells aresensitive to birinapant-induced death and that the clinical caspase inhibitor emricasan/IDN-6556 augments,rather than prevents, killing by birinapant. Deletion of caspase-8 sensitized AML to birinapant, whereas combinedloss of caspase-8 and the necroptosis effector MLKL (mixed lineage kinase domain-like) prevented birinapant/IDN-6556–induced death, showing that inhibition of caspase-8 sensitizes AML cells to birinapant-induced necroptosis.However, loss of MLKL alone did not prevent a caspase-dependent birinapant/IDN-6556–induced death, implyingthat AML will be less likely to acquire resistance to this drug combination. A therapeutic breakthrough in AML haseluded researchers for decades. Demonstrated antileukemic efficacy and safety of the birinapant/emricasancombination in vivo suggest that induction of necroptosis warrants clinical investigation as a therapeutic oppor-tunity in AML.

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INTRODUCTION

It is a hallmark of cancer cells that they become resistant to apoptosisas they acquire a malignant phenotype (1). This has led to the develop-ment of new therapies that resensitize cancers by targeting cell deathinhibitor proteins. For example, BH3 mimetics such as venetoclax an-tagonize prosurvival BCL-2 family proteins, whereas SMAC mimeticssuch as birinapant antagonize inhibitor of apoptosis proteins (IAPs)(2, 3). Both classes of compounds specifically inhibit protein-proteininteractions to activate cell death–inducing pathways.

SMAC mimetics were designed to antagonize X-linked IAP (XIAP)and relieve caspases from XIAP-dependent inhibition, thereby pro-moting cancer cell apoptosis (4). However, these compounds also bindto the BIR domains of cellular IAPs (cIAPs), promoting their auto-ubiquitylation and proteasomal degradation (5–7). cIAP degradationhas two consequences: in a subset of cancer cells, it activates a tran-scriptional pathway resulting in autocrine production of tumor necro-sis factor (TNF) (5, 6, 8), and it sensitizes cells to the cytotoxic activity

1Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3052, Australia2Department of Medical Biology, University of Melbourne, Melbourne, Victoria 3050Australia. 3Alfred Hospital and Monash University, Melbourne, Victoria 3004, Australia. 4SanfordBurnham Medical Research Institute, La Jolla, CA 92037, USA. 5Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NewSouth Wales 2031, Australia. 6Peter MacCallum Cancer Centre, Melbourne, Victoria3002, Australia. 7Burnet Institute, Melbourne, Victoria 3004, Australia. 8Departmenof Immunology, Central Clinical School, Monash University, Melbourne, Victoria 3004Australia. 9TetraLogic Pharmaceuticals Corporation, Malvern, PA 19355, USA. 10MurdochChildrens Research Institute, The Royal Children’s Hospital, Melbourne, Victoria 3052, Australia*These authors contributed equally to this work.†These authors contributed equally to this work.‡Corresponding author. Email: silke@wehi.edu.au (J.S.); paul.ekert@mcri.edu.au (P.G.E.

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of TNF, an activity usually kept in abeyance by the cIAPs (5, 9). Sev-eral studies have shown that IAPs also play a role in the regulation ofthe switch between two forms of cell death, apoptosis and necroptosis(10, 11). Upon TNF and TNF receptor 1 (TNFR1) interaction, a complex(complex I) containing TNFR-associated death domain (TRADD),receptor-interacting protein kinase 1 (RIPK1), TNFR-associated factor2 (TRAF2), and cIAP1/cIAP2 is recruited to the receptor. cIAPs pro-mote RIPK1 ubiquitylation contributing to canonical nuclear factor kBactivation. When cIAP1 and cIAP2 are depleted by SMAC mimetics,nonubiquitylated RIPK1 recruits caspase-8 to form an alternativecomplex, complex IIa, initiating apoptosis (12). In the presence of cas-pase inhibitors, a complex IIb is assembled, consisting of RIPK1, RIPK3,and MLKL (mixed lineage kinase domain-like), to induce programmednecrosis (necroptosis) (13). Thus, SMAC mimetic compounds can in-duce cell death via two pathways: a caspase-dependent apoptoticpathway and a caspase-independent necroptotic pathway.

Acute myeloid leukemia (AML) is an aggressive disease character-ized by low survival and high relapse rate (14–16), and new therapeu-tic strategies are urgently needed. AML is genetically heterogeneous,but certain chromosomal rearrangements occur commonly (17). Forexample, two subgroups of AML, infant AML and secondary AML inpatients previously treated with DNA topoisomerase II inhibitors,have a high frequency of 11q23 aberration and are associated with avery poor prognosis (18, 19). Rearrangements of 11q23 fuse the lysine(K)–specific methyltransferase 2A (KMT2A) gene encoding mixed-lineage leukemia (MLL) to 1 of at least 64 different partners. The trans-location partners MLLT3 (encoding AF9), MLLT1 (encoding ENL),and AFF4 (encoding AF4) are the most common translocations(20). These, and other translocation-driven leukemias, are modeled

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in mice using retroviral expression of the fusion protein in hemato-poietic stem cells (21–23).

We have developed clinically relevantmodels of AML to test in vitroand in vivo whether targeting IAPs with the clinical SMAC mimetic

birinapant is effective in the treatment ofAML and how it can be most effectivelyused to increase the chances of cure inAML. Using these models, we are ableto show that a clinical caspase-8 inhibi-tor, emricasan, can sensitize AML to bir-inapant and even overcomes birinapantresistance.

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RESULTS

AML cells harboring MLLtranslocation are sensitiveto birinapantTo determine the efficiency of birinapantin inducing apoptosis in AML cells, wetested four primary mouse AML modelsfor their sensitivity to birinapant (Fig. 1Aand fig. S1). In survival assays, MLL-AF9and MLL-ENL cells were sensitive andkilled by birinapant, whereas Nup98-HoxA9and HoxA9 + Meis1 leukemias were resist-ant (Fig. 1B). Cell death of MLL-ENL andMLL-AF9 was induced by 250 nM biri-napant, although 50 nM was sufficientto induced cIAP1 depletion (Fig. 1C). Thismay indicate that antagonism of XIAP isrequired for birinapant to induce cell deathbecause birinapant has a higher inhibitionconstant (Ki) against XIAP than cIAP1(24). Birinapant killed MLL-ENL cellsmore effectively than a standard AMLchemotherapeutic, cytarabine (ara-C) (Fig.1D). Leukemic cells pretreated with aTNFR1-blocking antibody were resistantto birinapant-induced cell death (Fig.1E), demonstrating that birinapant in-duced TNF/TNFR1-dependent cell deathin MLL-ENL leukemias.

Caspase inhibitors sensitize AMLcells to birinapantTo determine the mechanism of cell deathinduced by birinapant, AML cells weretreated with birinapant and the caspaseinhibitors Z-VAD-FMK and Q-VD-OPh(Fig. 2A). Unexpectedly, rather thaninhibiting birinapant-induced cell death,Z-VAD-FMK, but not Q-VD-OPh, sensi-tized MLL-ENL and MLL-AF9, but notHoxA9 + Meis1 cells, to birinapant killing(Fig. 2A). Caspase inhibitors might sensi-tize cells to SMAC mimetics by inhibiting

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caspase-8 andpromoting necroptotic cell death (Fig. 2B) (25–27). To testthis hypothesis, we generated MLL-ENL AML cells deficient in genesrequired for necroptosis (Fig. 2C and fig. S2). Deleting Tnfr1, Ripk3,orMlkl did not alter the onset of MLL-ENL leukemia, spleen weight,

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Fig. 1. AML driven by different oncogenes shows varying sensitivity to birinapant. (A) Schemat-ic of oncogenes and AML derivation protocol. Kaplan-Meier survival curve for mice transplanted with

cells transduced with the different oncogenes. GFP, green fluorescent protein; LTR, long terminalrepeat; IRES, internal ribosomal entry site; E14, embryonic day 14; rmIL-3, recombinant murineinterleukin-3; i.v., intravenously. (B) Survival of different AML cells in response to birinapant. Primaryleukemic cells from four AML models were treated with birinapant for 16 hours. Cell survival datarepresent means ± SEM of three to six independent tumors per model. PI, propidium iodide. (C) Pro-tein expression profile of MLL-ENL and MLL-AF9 cells 5 hours after birinapant treatment (0, 50, 100,or 250 nM). Data are representative of two independent experiments. WB, Western blot; cFLIP,cellular FLICE-like inhibitory protein. (D) Cell viability of MLL-ENL leukemic cells 16 hours after treat-ment with different concentrations of birinapant or ara-C. Means ± SEM, n = 6 independent tumors.(E) TNFR1 is required for cell death mediated by birinapant in MLL-ENL leukemias. Cells were treatedwith 100 nM birinapant ± TNFR1 antibody (10 mg/ml) for 16 hours. Data are means ± SEM, n = 3independent tumors.

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or white blood cell count on disease pre-sentation, showing that these genes do notplay a role in MLL-ENL–induced leuke-mogenesis (Fig. 2C and fig. S2). However,loss of Tnfr1 prevented birinapant-inducedand birinapant/Z-VAD-FMK (bir/ZVAD)–induced death (Fig. 2D and fig. S2E),whereas loss of Ripk3 or Mlkl alone didnot protect cells in short-term cell deathassays (Fig. 2D).

We then tested whether the sensitizingeffect of caspase inhibition functioned inbirinapant-resistant AML cells. To modelclinical drug resistance, we generatedMLL-ENL AML with acquired resistanceto birinapant (MLL-ENLR) by culturingMLL-ENL leukemic cells in increasingdoses of birinapant over 4 weeks (fig. S3A).The resultant AML was 10-fold more re-sistant than its founder lineor a line cultured

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Fig. 2. Caspase inhibition sensitizes leu-kemic cells to birinapant. (A) Cells from pri-

mary MLL-ENL, MLL-AF9, and HoxA9 + Meis1leukemias were pretreated for 15 to 30 minwith the caspase inhibitors Z-VAD-FMK (Z-VAD)(5 mM) or Q-VD-OPh (Q-VD) (10 mM) followedby 100 nM birinapant treatment for 16 hours,after which cell viability was determined. Dataare means ± SEM, n = 6. DMSO, dimethyl sul-foxide. (B) Schematic of TNF death signalingpathways associatedwith apoptosis and ne-croptosis. C8, caspase-8. (C) Kaplan-Meiersurvival curve of mice transplanted with MLL-ENL–transduced E14 liver cells of the indicatedgenotype. (D) MLL-ENL cells from wild-type(WT), Tnfr1−/−, Ripk3−/−, andMlkl−/−micewerepretreated (15 to 30 min) with the caspaseinhibitors Z-VAD-FMK (5 mM) or Q-VD-OPh(10 mM) followed by birinapant (100 nM). Cellviability was determined 16 hours after treat-ment. Means + SEM, n = 6. P values were ob-tained by comparing the survival of micewith WT and knockout leukemias treatedwith bir/ZVAD. (E) Birinapant-resistant MLL-ENL leukemic cells are sensitive to cell deathinduced by birinapant plus caspase inhibi-tors. Cells were pretreated (15 to 30min) withthe caspase inhibitors Z-VAD-FMK (10mM)orQ-VD-OPh (10 mM) followed by 100 or 500 nMbirinapant. Cell viability was determined16 hours after treatment. Data are means± SEM of two independent leukemias, eachtested in two independentexperiments (n=4).(F) Human AML cell line MV4-11 was treatedwith birinapant ± Z-VAD-FMK (10 mM) or Q-VD-OPh (10 mM). Data are means ± SEM, n =3. Cell survival was determined by propidiumiodide uptake and flow cytometry. *P< 0.005,***P < 0.0005, ****P ≤ 0.00005.

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without birinapant over 4 weeks but stillresponded to ara-C (fig. S3B). Westernblots of resistant and sensitiveAML treatedwith birinapant showed reduced caspaseactivation inMLL-ENLR cells when com-pared to sensitive and untreated cells (fig.S3C). Nevertheless, birinapant-resistantAML cells were strongly sensitive to biri-napant killing by Z-VAD-FMK or, to alesser extent, Q-VD-OPh (Fig. 2E). Thehuman AML cell line MV4-11 is naturallyresistant to birinapant but was also sensi-tive to Z-VAD-FMK, but not Q-VD-OPh,to a similar degree as the primary murineAML cells (Fig. 2, E and F).

The toxicity of Z-VAD-FMK preventsits clinical use (28, 29), and Q-VD-OPh

was unable to synergize with birinapant to kill AML cells. Therefore, tosee whether this finding might be translatable into the clinic, we testedwhether birinapant in combination with a clinical caspase inhibitor

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could kill AML cells. IDN-6556/emricasan is a caspase inhibitor thatinhibits apoptosis in the liver and is well tolerated in humans (30).Emricasan was better at promoting birinapant-induced cell death than

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Fig. 3. Caspase-8 inhibitors are potent in-ducers of TNF-RIPK1–dependent cell death in AML cells. (A) MV4-11 cells were pretreated(15 to 30 min) or not with the caspase inhib-itor IDN-6556 (5 mM) followed by birinapant atthe specified doses for 48 hours. Data aremean ± SEM, n = 5. (B) MLL-ENL birinapant-sensitive (S) and MLL-ENL birinapant-resistant(R) leukemic cells were pretreated (15 to 30min)with IDN-6556 (5 mM) or vehicle followed bybirinapant (100 nM) or vehicle. Cell viabilitywas determined after 16 hours. Means ± SEM,n = 5. (C) Survival of MLL-AF9, HoxA9 + Meis1,and Nup98-HoxA9–transformed AML cells.Cells were pretreated with the caspase inhib-itors IDN-6556 (5 mM) or Q-VD-OPH (10 mM)followed by birinapant (100 nM for MLL-AF9cells and 500 nM for HoxA9 + Meis1 andNup98-HoxA9) ± Nec-1 (50 mM) for 16 hours.(D to F) TNF concentration in total cell lysatesof MLL-ENL birinapant-sensitive and birinapant-resistant, MLL-AF9, and HoxA9 +Meis1 cells wasdetermined after birinapant (100 nM for MLL-ENL and MLL-AF9 and 500 nM for HoxA9 +Meis1), IDN-6556 (5 mM), or bir/IDN treat-ments for the indicated time period. Data aremeans ± SEM (n = 4 to 9) of three indepen-dent experiments. (G to I) TNF concentrationsin total cell lysates of MLL-ENL birinapant-sensitive (S) and birinapant-resistant (R), MLL-AF9, andHoxA9+Meis1 cellswere determinedafter 9 hours of treatment with bir/IDN ±Nec-1 (50 mM). Drug concentrations were thesame as in (D) to (F). Data are means ± SEM,n = 3 to 4. P value was determined by com-parison of bir/IDN and bir/IDN + Nec-1 for eachleukemiasubtype.Cell survivalwasdeterminedby propidium iodide uptake and flow cytom-etry. *P < 0.05, **P < 0.005, ***P < 0.0005.

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either Z-VAD-FMKorQ-VD-OPh (compare Fig. 2, E and F, with Fig. 3,A and B, and fig. S3, D to F), and very few AML cells from differentmodels survived the combined birinapant/IDN-6556 (bir/IDN) treat-ment (Fig. 3C and figs. S3E and S4, A and B). From a clinical perspec-tive, MLL-ENL cells, with acquired resistance to birinapant (Fig. 3Band fig. S3, D and E) or ara-C (fig. S4A) or which coexpress an activatingRas mutation (fig. S4B) and are therefore resistant to standard chemo-therapy (23, 31), were all equally sensitized to birinapant killing byIDN-6556 (Fig. 3B and figs. S3, D and E, and S4, A and B). However,AML-ETO9a and CBFb/MYH11 cells with the same active Ras mu-tation could not be killed even by high concentrations of bir/IDN (fig.S4, B and C). In a subset of cancers, SMACmimetics cause cell death bysimultaneously inducing TNF and sensitizing cells to TNF-induced celldeath (5–9). If cells fail to produce TNF, they can often be sensitized tokilling by exogenously adding TNF. However, CBFb/MYH11 cells werecompletely resistant, and AML-ETO9a cells were relatively resistant tothe birinapant/emricasan/TNF cell death stimulus (fig. S4C), suggestingthat resistance to the combination occurs downstreamof TNFR1. Thereis, however, no obvious correlationwith the expression of RIPK1, RIPK3,andMLKL, which are present in sensitive and resistant cell lines alike(figs. S3F and S4D). Finally, bir/IDNwas able to killBax−/−Bak−/− cellsthat are completely resistant to intrinsic cell death stimuli such asmightbe mediated by cytotoxic drugs like ara-C (fig. S4E).

IDN-6556 combines with birinapant to induce TNFexpression and necroptosis in AMLWenext testedwhether IDN-6556 sensitized leukemic cells to birinapantby increasing TNF production.When treated with the bir/IDN com-bination, but not with either compound alone, MLL-ENL birinapant-sensitive and birinapant-resistant leukemias, as well as MLL-AF9 andHoxA9 +Meis1 leukemias, rapidly induced detectable amounts of TNF(Fig. 3, D to F). The kinase activity of RIPK1 plays a role in SMACmimetic–induced TNF production in some cell types (11, 32). There-fore, to determine the role of RIPK1 kinase activity in TNF productionin our leukemic models, we treated MLL-ENL, MLL-AF9, andHoxA9 + Meis1 cells with bir/IDN in the presence or absence ofthe RIPK1 kinase inhibitor necrostatin (Nec-1). The addition ofNec-1 strongly reduced TNF production and thereby blocked celldeath in both murine and human AML cells (Fig. 3, C, G, and I,and figs. S3E and S5, A and B). Genetic deletion of Tnfr1 or TNFR1inhibition alsopreventedbir/IDNkilling inmouse andhuman leukemias,thus confirming that the fundamentalmechanismof killingby birinapantand bir/IDN is the same in these cells (Fig. 4A and fig. S5, C and D).

Because Nec-1 prevented TNF production by bir/IDN, its use didnot address whether AML cells treated with bir/IDN died by necroptosis.To determine whether bir/IDN induced necroptosis, we generatedMLL-ENL leukemias lacking necroptosis effector genes. Ripk3 andMlkl deficiency did not affect bir/IDN-induced TNF production(Fig. 4B). Tnfr1 deficiency also did not entirely prevent bir/IDN-inducedTNF production, showing that it is an intrinsic property of the bir/IDN combination rather than cell death driving TNF production(Fig. 4B). Although Ripk3 andMlkl deficiency did not affect TNF pro-duction, they did reduce bir/IDN-induced cell death in short-term celldeath assays (graphs, Fig. 4A). However, in long-term clonogenic as-says, neither Ripk3 norMlkl deficiency provided any protection (plates,Fig. 4A). On the other hand, Casp8−/−Ripk3−/− and Casp8−/−Mlkl−/−

MLL-ENL leukemias were completely resistant to bir/IDN treatment(Fig. 4A and fig. S6, A to C), suggesting that residual caspase-8 activity

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was responsible for the cell death observed in Ripk3−/− and Mlkl−/−

cells treated with bir/ZVAD or bir/IDN. On the basis of these results,we made two predictions: first, a comparison of short- and long-termdeath assays suggested that among necroptosis-deficient cells, bir/IDN-treated cells die more slowly than wild-type cells, and second,the genetic results indicated that the slower cell death was caspase-dependent and therefore might be blockable by an additional caspaseinhibitor. Consistent with these predictions, cell death mediated bybir/IDNwas delayed in Ripk3−/− orMlkl−/− compared to wild-type cells(Fig. 4C), whereas high-dose Q-VD-OPh or readdition of IDN-6556prevented bir/IDN-induced cell death inRipk3−/− andMlkl−/−butnot inwild-type AML (Fig. 4, D and E). Moreover, cleaved caspase-8 andcaspase-3 were present inMlkl−/− and Ripk3−/− MLL-ENL leukemiastreated with bir/IDN (fig. S6D). Together, these results suggest thatIDN-6556 is effective at inducing necroptosis in birinapant-treatedAML cells, by simultaneously increasing TNF production and blockingapoptosis. However, possibly because of the increased TNF production,its activity is ultimately insufficient to block all caspase-8 activity, there-by causing cells that are unable to die by necroptosis to die by apoptosis.These results also imply that loss of caspase-8 should be sufficient tosensitize AML cells to birinapant.

Caspase-8/cFLIPL is the key target ofbir/IDN-mediated necroptosisWe tried several different approaches to deleteCasp8 from the fetal liverprogenitors or the AML cells themselves, but this always resulted in celldeath. Finally, we were able to generate caspase-8–deficient cells by re-expressing inducible MLKL (MLKLi) in Casp8−/−Mlkl−/− MLL-ENL leu-kemias (fig. S6E). Consistent with our hypothesis that loss of caspase-8would be sufficient to sensitize these cells, birinapant induced cell deathin Casp8−/−Mlkl−/− MLKLi AML cells to a level comparable to that inwild-type AML cells treated with bir/IDN (compare Fig. 4F to Fig. 4A).

On the basis of these observations, we suspected that the differentialability of IDN-6556, Z-VAD-FMK, and Q-VD-OPh to sensitize AMLto birinapant-induced cell death may correlate with their respectiveability to inhibit caspase-8. The caspase-8/cFLIPL heterodimer is cata-lytically active and has been proposed to be the form of caspase-8 thatinhibits necroptosis (33, 34).We therefore compared the ability of thesecaspase inhibitors to inhibit the caspase-8 homodimer or the caspase-8/cFLIPL heterodimer. We generated caspase-8 homodimers or caspase-8/cFLIPL heterodimers in vitro as described (33) and incubated themwith afluorogenic substrate in the presence of each inhibitor. Inhibition rateconstant (kobs/I) values for both protease species show that IDN-6556is a better caspase-8/cFLIPL heterodimer inhibitor than either Z-VAD-FMK or Q-VD-OPh (Fig. 4G and fig. S7). Moreover, IDN-6556 andZ-VAD-FMK inhibited the caspase-8/cFLIPL heterodimermore efficient-ly than Q-VD-OPh. These results fit a model whereby inhibition of thecaspase-8/cFLIPL heterodimer efficiently activates birinapant-inducednecroptosis (fig. S8, A and B). However, neither Z-VAD-FMK nor IDN-6556canrestrainhomodimeric caspase-8 sufficiently topreventbirinapant-induced apoptosis in necroptosis-deficient cells (Figs. 2D and 4, C to E).IDN-6556, like Q-VD-OPh and Z-VAD-FMK, also inhibits the apo-ptotic caspase-3 and caspase-9 in vitro (fig. S7). However, in contrastto their potential to synergize with birinapant, Q-VD-OPh inhibitedara-C–induced apoptosis of MLL-ENL leukemic cells in a dose-dependent manner, whereas IDN-6556 at the same concentrations wascompletely ineffective at inhibiting the intrinsic apoptotic pathway (fig.S8C). These contrasting results in the same cells show that it is the

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Fig. 4. IDN-6556 inhibits the caspase-8/cFLIPL heterodimer to induce necroptosis in

birinapant-treated leukemias. (A) MLL-ENLleukemias derived from WT, Tnfr1−/−, Ripk3−/−,Mlkl−/−, Casp8−/−Ripk3−/−, and Casp8−/−Mlkl−/−

progenitors were pretreated with IDN-6556(5 mM) or vehicle followed by birinapant (50 nM)or vehicle. Graphs: Cell viability was determined16 hours after treatment with propidium iodidestaining and flow cytometry. Mean ± SEM, n = 6.Images: Clonogenic assay to determine long-termsurvival and proliferation of MLL-ENL leukemiacells treated with birinapant (250 nM) ± IDN-6556 (5mM).Cellswerepretreated for 30minwithIDN-6556 followed by birinapant. After the pre-treatment, 500 cells were plated in 0.3% agarcontaining birinapant or bir/IDN. Clonogenic sur-vival was determined 15 days after treatment.(B) TNF production inWT, Tnfr1−/−, Ripk3−/−, andMlkl−/−MLL-ENL leukemias. Cells were treatedwith birinapant (100 nM) ± IDN-6556 (5 mM) for9hours, and TNF concentrationsweredeterminedinwhole-cell lysates by enzyme-linked immuno-sorbent assay (ELISA). Mean ± SEM, n = 5. P valueswere obtained by comparison of birinapant-and bir/IDN-treated leukemic cells from eachgenotype. (C) Time course of killing of MLL-ENLleukemias derived from WT, Mlkl−/−, and Ripk3−/−

progenitors pretreated with IDN-6556 (5 mM)followed by birinapant (100 nM). P values werecalculated by comparing WT leukemic cells withthe specific knockout ateach timepoint: 8, 10, and16 hours. (D) MLL-ENL leukemias derived fromWT, Mlkl−/−, and Ripk3−/− progenitors were pre-treatedwith IDN-6556 (5 mM) followed by birina-pant (100 nM) ± Q-VD-OPH (10mM). (E)MLL-ENLleukemias derived from WT, Mlkl−/−, and Ripk3−/−

progenitors were pretreated with IDN-6556 (5 mM)followed by birinapant (100 nM). IDN-6556 wasreadded to cells 3 hours after birinapant treat-ment, and cell survival was determined 16 to 18hours later. Data are means ± SEM, n = 4. (F)MLL-ENL Casp8−/−Mlkl−/− leukemia cells weretransduced with a lentiviral construct expressingMLKL from a doxycycline (dox)–inducible promoter.Leukemic cells were treated with birinapant ±IDN-6556 in the presence or absence of doxycy-cline (0.5 mg/ml) to induce MLKL. Cell viability wasdetermined 16 hours after treatment. Mean ±SEM, n = 4. (G) Comparison of the ability ofZ-VAD-FMK, Q-VD-OPH, and IDN-6556 to inhibitrecombinant caspase-8 homodimer and caspase-8/cFLIP heterodimer. Values are means ± SD ofduplicate experiments. Cell survival was deter-mined by propidium iodide uptake and flow cy-tometry. *P < 0.05, **P < 0.005, ***P < 0.0005.

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preferred caspase-8/cFLIP target of these inhibi-tors rather than their drug-like properties, suchas cell permeability, that determines whether ornot they synergize with birinapant to kill AMLcells.

Necroptosis mediated by bir/IDNeliminates AML in vivoTo determine whether the combination of bir/IDN was tolerated and effective in vivo, wetransplanted mice with MLL-ENL cells bearinga luciferase transgene. Dosing commenced whenthe disease burden in these animals reached an

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Fig. 5. Combination of birinapant and the clinicalcaspase-8 inhibitor emricasan/IDN-6556 prolongs

survival in AML models. (A) In vivo imaging of tu-mor progression in mice treated with vehicle (6%Captisol in phosphate-buffered saline), IDN-6556(2.5 mg/kg), birinapant (5 mg/kg), or the combina-tion. Mice were treated with the drugs twice aweek for 4 weeks, starting treatment 10 to 11 daysafter retransplant of tumor cells. Images are repre-sentative of animals from each treatment group atthe specified time point. i.p., intraperitoneal. (B) Graphsrepresent average photons per second valuesfrom mice treated with birinapant, IDN-6556, orthe combination. Error bars represent SEM of n =14 for vehicle, n = 10 for IDN-6556, n = 14 for birina-pant, and n = 16 for bir/IDN. (C) Kaplan-Meier sur-vival curve of albino C57BL/6 mice transplantedwith MLL-ENL leukemia and treated with vehicle,birinapant, IDN-6556, or the combination (bir/IDN). Arrows indicate the start and end of treat-ment schedule. Drug concentrations used as de-scribed in (A). Statistical analysis was performed bycomparison of birinapant or bir/IDN-treated micewith vehicle (blue) or IDN-6556 (orange). n.s., notsignificant. (D) Hematoxylin and eosin–stainedsections of spleen and liver from mice with MLL-ENL AML after treatment with IDN-6556, birina-pant, or the combined therapy. Arrows indicateinfiltration of blast leukemic cells. Scale bars,200 mm for the (spleen images) and 100 mm (liverimages). (E) bir/IDN treatment is effective in ALL(acute lymphoblastic leukemia) and AML PDX samples.Median inhibitory concentration (IC50) of birina-pant or bir/IDN treatment was determined forthese PDX samples. Cells were treated for 48 hourswith birinapant (10 to 1000 nM) ± IDN-6556 (5 mM),and cell viability was determined by annexin V/7-AAD staining. Data are means of four independentexperiments. N/A, not applicable. (F) Combinedtherapy kills patient leukemia samples. Primary leu-kemic cells derived frompatientswith the indicatedtreatment statuswere treated for 24 hours with bir-inapant (500 nM) ± IDN-6556 (5 mM). Cell viabilitywas determined by flow cytometry of CD34+ propi-dium iodide–negative cells. Data are means of twoindependent experiments. *P < 0.05, **P < 0.005.hCD34, human CD34.

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emission of 107 to 108 photons/s (Fig. 5, A and B). bir/IDN treatment waswell tolerated in vivo (fig. S9A), and it reduced MLL-ENL disease bur-den and increased long-term survival inmost of the animals, with sur-vival extending 80 to 100 days after the treatment began (Fig. 5, B andC). The combination treatment was also effective at prolonging sur-vival in mice bearing either MLL-AF9 or MLL-ENL birinapant-resistantAML cells (fig. S9, B and C). A smaller proportion of animals re-sponded to birinapant alone, whereas untreated mice and those receiv-ing IDN-6556 alone rapidly succumbed to AML. Histological examinationconfirmed remission in combination-treated animals (Fig. 5D). IDN-6556 accumulates in the liver (28), and thus, as predicted, combina-tion-treatedmice had fewer leukemic cells in the liver than those treatedwith birinapant (Fig. 5D). The same thing was observed in the spleen ofcombined therapy mice because the bir/IDN-treated mice had anincreased number of splenic follicles, indicating less infiltration of blastcells in the organ.

The tolerability and efficacy of bir/IDN therapy wereevaluated in human primary cellsFinally, we tested the bir/IDN combination in primary human hema-topoietic cells, patient-derived xenografts (PDXs) from MLL-harboringAML and ALL, and bone marrow and peripheral blood samples frompatients with AML (Fig. 5, E and F, and fig. S10). The combined treat-ment was less toxic to CD34+ stem cells (fig. S10, A and B), peripheralblood mononuclear cells, T cells, B cells, and natural killer cells thanstandard chemotherapy (fig. S10C) and effective in three of four PDXsamples tested (Fig. 5E). To extend our analysis, we determined theresponse of AML cells from bone marrow and peripheral blood sam-ples to birinapant or bir/IDN treatment (Fig. 5F and table S1). Overall,four of eight samples were sensitized to birinapant killing by IDN-6556 (patients 1, 2, 3, and 6). Some chemoresistant AML cells frompatients with relapsed or secondary disease were among those thatwere sensitive to bir/IDN (patients 1 and 2; Fig. 5F). Four otherAML samples responded to birinapant alone (4, 5, 7, 8), and in threeof these, birinapant-induced cell death was inhibited by the addition ofIDN-6556. These results show that combined IAP/caspase-8/cFLIPLinhibition may be a potential therapeutic combination for many pa-tients, especially ones who have failed standard chemotherapy.

2020

DISCUSSION

Despitemore than three decades of research, the survival rates inAMLhave only improved by 24%, with more than 40% of the patients re-lapsing within 12 months after intensive chemotherapy (15). In mostAML cases, chemoresistance is strongly associated with increased ex-pression of antiapoptotic proteins, such as BCL-2 and XIAP, and fail-ure of leukemic cells to undergo apoptosis (2, 35–37). Therefore, newtherapies are more likely to succeed if the proposed mechanism ofaction is distinct from factors typically linked to chemoresistance.

SMAC mimetics such as birinapant are in clinical trials, but likeother targeted anticancer drugs, they have limited effectiveness assingle agents. Here, we used two clinically relevant compounds andshowed that the combination of birinapant plus the clinical caspaseinhibitor emricasan/IDN-6556 is well tolerated and efficiently killsAML cells in vitro and in vivo by activation of the necroptotic pathway.Together, these findings validate the potential of necroptosis targetingas a therapeutic strategy to overcome chemoresistance and treat AML.

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In our in vitro assays, we showed that AML subtypes from murinemodels and human patient cells differ in their sensitivity to birinapanttreatment. However, addition of caspase inhibitors, and particularlyemricasan, strongly sensitizedmanyAML types thatwe tested to birinapant-induced cell death. Even AML cells with natural or acquired resistancetobirinapant, such asmight occur clinically,were killedby the combinationofbirinapantplus caspase inhibitors. Because caspase inhibition in thepres-ence of an IAP antagonist (birinapant) can cause necroptosis, this result im-plies that activation of necroptosis is a means to overcome chemoresistance.

Previous studies have shown that necroptosis induced by an experi-mental SMACmimetic, BV6, and an experimental caspase inhibitor,Z-VAD-FMK, can sensitize AML cell lines to chemotherapy (36, 38, 39).Our studies extend this concept. We showed that the combination of aclinical SMACmimetic and a clinical caspase inhibitor can inducenecrop-tosis without the addition of chemotherapy and more effectively than theexperimental compounds. Furthermore, this combination was well toler-ated and effective in vivo, despite the pharmacodynamic limitations ofemricasan. The inhibitory profile of emricasan strongly suggests that thereason for themuch greater efficacy of this compound compared toQ-VD-OPh in cooperating with birinapant is its ability to inhibit the caspase-8/cFLIPLheterodimer. It hasbeen suspected that the caspase-8/cFLIPLhetero-dimer is particularly important for inhibiting necroptosis, and the fact thatemricasan is so effective at inducing necroptosis whereas Q-VD-OPhis not strongly supports this notion (33, 34). Our genetic experimentsalso revealed an unexpected twist:Casp8−/−Ripk3−/− andCasp8−/−Mlkl−/−

AML cells are completely resistant to birinapant/emricasan-induceddeath, but Ripk3−/− andMlkl−/− AML cells are completely sensitive in long-term survival assays. In short-term survival assays, this death occursmoreslowly than inwild-type cells and canbe blockedby an additionofQ-VD-OPh. These data indicate that although birinapant and emricasan arestrong activators of necroptotic cell death, they can also nevertheless in-duce an apoptotic cell death. This unusual activity seems to be due to theability of emricasan to increase birinapant-induced TNF production, andthis is even the casewhen cells are selected for resistance to birinapant. Themechanism for this ability to increase TNF production requires RIPK1kinase activity because Nec-1 prevents the bir/IDN-induced TNF increase.However, unlike birinapant-induced TNF in bone marrow–derivedmacrophages (11), it is unlikely that it requires RIPK3 because Ripk3−/−

AML cells were still sensitive to the bir/IDN combination.Although our results hold great promise for translation, emricasan

is not a perfect drug for AML treatment because it has a half-life ofonly 50 min in plasma, resulting from a first-pass effect in the liver(30). The strong inhibition of AML disease in the liver that we observedwith birinapant/emricasan is entirely consistent with these pharmaco-kinetics. It is clear, however, that for caspase inhibition to be used in theclinic together with birinapant, either emricasan will have to be usedwith an alternative dosing schedule or a caspase inhibitor with the sameinhibitory profile as emricasan but better pharmacokinetics will needto be developed. At the very least, our work should reinvigorate inter-est in developing such inhibitors with a focus on developing drugs thatinhibit the caspase-8/cFLIPL heterodimer.

MATERIALS AND METHODS

Study designIn our hypothesis-driven experimental design, we addressed the thera-peutic potential of the clinically relevant drug combination bir/IDN in

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human AML cell lines, primary human AML cells, and primary murineAML cells. The effect of the combination therapy on cell death signaling(apoptosis and necroptosis) and cell growth was assessed by flow cytom-etry analysis of cell death markers and colony formation assays. Thisstudy was extended to a murine model of AML to analyze the effectsof combination therapy in vivo. Tolerability and biosafety of the com-bined therapy bir/IDN were tested in vivo in healthy murine specimensand in vitro using CD34+ stem cells and blood cells from healthy pa-tients. All in vivo experiments were performed in 8- to 10-week-oldmice housed in individually ventilated cages and following the Walterand Eliza Hall Institute (WEHI) Animal Ethics Committee (AEC) re-commendations for animal welfare and ethical approach to animals inexperimental procedures. In vivo experiments were performed twoto three times, and they were conducted and analyzed in a blindedmanner. Details on replicates and statistical analyses of in vivo andin vitro experiments are indicated in the figure legends.

ReagentsInhibitors Z-VAD-FMK and Q-VD-OPH and fluorescent substrateAc-IETD-afc were purchased from SM Biochemicals. InhibitorIDN-6556 and A/C heterodimerizer used in biochemical assays werepurchased from MedChem Express LLC and Clontech, respectively.Fluorescence measurements were performed in 96-well white platesusing the Gemini EM fluorometer fromMolecular Devices. The SMACmimetic birinapant and the caspase-8 inhibitor IDN-6556 used for invitro and in vivo experiments with AML cells were provided by Tetra-Logic Pharmaceuticals. Ara-C and daunorubicinwere purchased fromSelleckchem Pharma. Blocking TNF antibodies, murine anti-TNFR1,and human anti-TNFa were purchased from R&D Systems.

Generation and culture of murine AML cellsAll in vivo experiments were conducted in accordance with the WEHIAEC guidelines. MLL-ENL, MLL-AF9, HoxA9 + Meis1, and Nup98-HoxA9 retroviral constructs were previously described (40–43). Viralsupernatants were produced in 293T cells by cotransfection of oncogeneconstructs and packaging plasmids. Progenitor/stem cells derived fromfetal liver (E14.5) of C57BL/6 Ly5.2 wild-type, Tnfr1−/−, Ripk3−/−,Mlkl−/−, Casp8−/−Mlkl−/−, Casp8−/−Ripk3−/−, and Bax−/−Bak−/− mice(44, 45) were cultured in a–minimum essential medium (a-MEM)(Invitrogen) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, murine stem cell factor (SCF) (100 ng/ml) (PeproTech), IL-6(10ng/ml), thrombopoietin (50 ng/ml), and FMS-related tyrosine kinase3 (FLT) (10 ng/ml) (all produced at WEHI) and transduced with viralsupernatant for two consecutive days using the RetroNectin protocol.RetroNectin (30 mg/ml; WEHI)–precoated 24-well plates were spunwith viral supernatant at 3000 rpm for 1 hour at 30°C and then seededwith 1 × 106 to 1.5 × 106 cells per well in supplemented a-MEM. Afterinfection, transduced cells were injected into sublethally g-irradiated(7.5Gy) C57BL/6 Ly5.1mice. Leukemia development was evidenced byweight loss, enlarged spleen, anemia, lethargy, and hunched posture.Leukemic cells were obtained from the bone marrow of sick miceand cultured at 37°C in a 10%CO2–humidified atmosphere in Iscove’smodified Dulbecco’s medium supplemented with 10% FCS and IL-3(3 ng/ml; R&D).

Clonogenic assaysColony formation assays for AML cells were performed as previouslydescribed (46).

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Human leukemic cellsHuman leukemic cell lines MV4-11 and U937 were cultured at 37°C in a10% CO2–humidified atmosphere in RPMI medium supplementedwith 10% FCS. Patient-derived leukemic cells were cultured in Stem-Span serum-free expansion medium II (STEMCELL Technologies)supplemented with recombinant human cytokines rhFlt-3L (100 ng/ml), rhSCF (100 ng/ml), rhIL-3 (20 ng/ml) and rhIL-6 (20 ng/ml)(R&D), deoxyribonuclease I (100 IU/ml) (Roche), and penicillin/streptomycin (Gibco) in 10% CO2 at 37°C.

Western blots and reagentsTotal cell lysates were prepared by lysing 1 × 107 cells/ml in SDSbuffer [50 mM tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 2.5%b-mercaptoethanol] and boiling for 10 min. The antibodies used wereanti-mouse caspase-8 polyclonal antibody (pAb), anti–cleaved caspase-8(Asp387) pAb, and anti–cleaved caspase-3 (Asp175) pAb (Cell Signaling);anti-cFLIP (Dave-2, Adipogen) monoclonal antibody (mAb); anti-RIPK3 pAb (ProSci); anti-RIPK1 mAb (BD); anti-cIAP1 mAb (Enzo);and anti-actin mAb (Sigma).

TNF ELISALeukemic cells from different AML models were pretreated for 30 minwith IDN-6556 followed by 100 nM birinapant. Cell lysates wereprepared by lysing 1 × 106 cells in 50 ml of ice-cold protein DISC lysisbuffer [150 mM sodium chloride, 2 mM EDTA, 1% Triton X-100,10% glycerol, 20 mM tris (pH 7.5)]. TNF content in whole-cell lysateswas measured by ELISA according to the manufacturer’s instructions(eBioscience).

Protein expression, purification, and nomenclatureGenes for human His-tagged caspase-8 (DCARD) and FLIPL (DDEDs)were expressed in Escherichia coli BL21 (DE3). Protein production wasinduced with 1 mM isopropyl-b-D-1-thiogalactopyranoside, and pro-teins were purified by Ni2+ affinity chromatography. For caspase-8, af-finity chromatography was followed by further purification on a MonoQ anion exchange Sepharose column using the ÄKTA system. Genera-tion of the caspase-8 homodimer (CASP8/CASP8) and the caspase-8/cFLIPL heterodimer (CASP8/FLIP) has been described elsewhere (33).Briefly, the caspase-8monomer forming theCASP8/CASP8 dimer con-sists of thewild-type sequence of the large and small subunits. Twoaminoacids, Asp374 and Asp384, located in the linker between the large and smallsubunits, were mutated to Ala to prevent autocleavage in the caspase-8that formsCASP8/FLIP.This double-mutant caspase-8wasC-terminallyfused to the dimerization domain FKBP, rendering the working speciesFKBP-CASP8D2A.

Dimer activation and kinetic characterizationActivity of commercial inhibitor preparations was determined by backtitration with caspase-3 using 200 mMAc-IETD-afc and different dilu-tions of the inhibitors. Residual enzymatic activity was plotted againstthe concentration of inhibitor. In reactions with CASP8/CASP8 pro-teins, inhibitors and substrate were prepared in a kosmotropic solution[30 mM tris-HCl (pH 7.4), 1 M sodium citrate, 5 mM dithiotreitol(DTT), and 0.05% CHAPS]. Reactions with CASP8/FLIP proteins, in-hibitors, and substrate were prepared in caspase buffer [20 mM Pipes(pH 7.2), 0.1 M NaCl, 1 mM EDTA, 10% sucrose, 0.1% CHAPS, and5 mMDTT]. Activation of the CASP8/CASP8 homodimer was achievedby incubating caspase-8 at a final concentration of 10 nM in kosmotropic

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solution for 20 min at 37°C. Upon activation, the homodimer was re-acted in 100 ml with a mixture of 200 mM Ac-IETD-afc and inhibitorat different concentrations. Reaction progress was monitored by fluo-rescence emission of the Ac-IETD-afc group every 2 s for 15 min at37°C. To activate the CASP8/FLIP heterodimer FKBP-CASP8D2A ata final concentration of 50 nM, FRB-FLIP at 250 nM and A/C hetero-dimerizer at 250 nM were incubated in caspase buffer for 20 min at37°C. Upon activation, the heterodimer was reacted in 100 ml with amixture of 200 mM Ac-IETD-afc and inhibitor at different concentra-tions. Reaction progress was monitored by fluorescence emission ofthe Ac-IETD-afc group every 2 s for 15 min at 37°C. Michaelis con-stant (Km) of CASP8/CASP8 and CASP8/FLIP for Ac-IETD-afc wasdetermined by activating each species with the protocol describedabove but with the substrate reacted in a concentration gradient from1 to 128 nM for the homodimer and 10 to 320 nM for the heterodimer.Reaction progress was monitored by fluorescence emission of the Ac-IETD-afc group every 2 s for 15 min at 37°C.

Data analysis and statisticsData analysis of biochemical caspase assays was performed with Prism6.0 software. Calculations were performed with the linear segment ofeach progress curve (approximately the first 200 s).

kobs/I of Z-VAD-FMK, Q-VD-OPH, and IDN-6556 for CASP8/CASP8 was obtained by fitting reaction rates to the pseudo–first-ordermodel:

Vobs ¼ Vs*½S� þ ðV0 � VsÞ�1� expð�kobs*½S�Þ

�=kobs

where S is the substrate concentration, V0 is the initial velocity, and Vs isthe steady-state rate of substrate hydrolysis in the presence of inhibitor. Toobtain kobs/I of Q-VD-OPH and IDN-6556 for CASP8/FLIP, we appliedthe same model, but for Z-VAD-FMK, a hyperbolic inhibition curve wasobserved, and k2/KI was calculated instead. To derive k2/KI, rates werefitted to a hyperbola using the following model:

k2=KI ¼ k2*½E�=ðKI þ ½E�ÞKm of CASP8/CASP8 and CASP8/FLIP for Ac-IETD-afc was

calculated using the following equation:

Vobs ¼ ½E�*kcat*½S�=ðKm þ ½S�ÞStatistical analysis of leukemic cells in vitro and in vivo was per-

formed with Prism 6.0 software. All experiments with n≥ 3 are rep-resented as means ± SEM. Data from in vitro assays were analyzedusing Student’s t test (two-tailed), and Kaplan-Meier survival curveswere analyzed using the log-rank (Mantel-Cox) test and log-rank testfor trend. P values are indicated in the figure legends, considering P <0.05 significant.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/339/339ra69/DC1Fig. S1. Endogenous protein expression in different AML subtypes.Fig. S2. Generation of single-gene knockout leukemias.Fig. S3. Generation of MLL-ENL birinapant-resistant cells.Fig. S4. IDN-6556–mediated TNF-dependent necroptosis in different AML subtypes.Fig. S5. Prevention of IDN-6556–induced cell death in human AML cells by inhibiting TNFR1 orRIPK1 kinase activity.Fig. S6. Generation of MLL-ENL double-gene knockout leukemias.Fig. S7. Biochemical comparison of caspase inhibitors IDN-6556, Z-VAD-FMK, and Q-VD-OPh.

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Fig. S8. Comparison of the ability of caspase inhibitors to induce or block cell death inleukemic cells.Fig. S9. Combined treatment with bir/IDN in vivo.Fig. S10. Safety of combined bir/IDN treatment in healthy human cells.Table S1. Deidentified patient data.

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Acknowledgments: We thank the staff of the WEHI Bioservices facilities and the Burnet In-stitute ImmunoMonitoring Facility for technical assistance. We also thank W. Alexander forproviding the Mlkl−/− and Casp8−/−Mlkl−/− mice and G. Begley and K. Manson for discussionsand intellectual input. Funding: This work was supported by a grant from the Leukemia & Lym-phoma Society [SCOR (Specialized Center of Research) grant 7001-06 to D.L.V.], the NationalHealth and Medical Research Council (NHMRC; grants 461221, 1025594, 1046010, and1081376), NHMRC fellowships (grants 541901 and 1058190 to J.S.), and an Association pourle Recherche contre le Cancer (ARC) fellowship to N.L. with additional support from the Austra-lian Cancer Research Foundation, the Victorian State Government Operational InfrastructureSupport, and the NHMRC Independent Research Institutes Infrastructure Support Scheme (grant361646). G.S. and M.N. are supported by NIH (grants RO1GM099040 and 5 R01CA163743). R.L. issupported by an NHMRC fellowship (1059804). Author contributions: G.B., C.M., N.L., N.-Y.N., M.N.,M.C.T., J.R., M.G., J.M.S., N.S., G.P., E.d.V., and R.G. designed and performed the experiments andanalyzed the data; G.B., J.S., P.G.E., D.L.V., A.W., and G.S. planned the project, designed theexperiments, and analyzed the data; S.P.G., S.M.C., R.W.J., R.L., and M.A.G. contributed exper-tise and experimental reagents; and G.B., J.S., and P.G.E. wrote the paper. Competing interests: J.S.and D.V. are on the scientific advisory board of TetraLogic Pharmaceuticals Corporation and holdoptions on a small number of shares in the company. G.S. serves as a consultant for Genentech. G.B.and J.S. have a provisional patent application related to the subject matter of the paper.

Submitted 26 August 2015Accepted 4 April 2016Published 18 May 201610.1126/scitranslmed.aad3099

Citation: G. Brumatti, C. Ma, N. Lalaoui, N.-Y. Nguyen, M. Navarro, M. C. Tanzer, J. Richmond,M. Ghisi, J. M. Salmon, N. Silke, G. Pomilio, S. P. Glaser, E. de Valle, R. Gugasyan,M. A. Gurthridge, S. M. Condon, R. W. Johnstone, R. Lock, G. Salvesen, A. Wei, D. L. Vaux,P. G. Ekert, J. Silke, The caspase-8 inhibitor emricasan combines with the SMAC mimeticbirinapant to induce necroptosis and treat acute myeloid leukemia. Sci. Transl. Med. 8,339ra69 (2016).

nceTranslationalMedicine.org 18 May 2016 Vol 8 Issue 339 339ra69 11

necroptosis and treat acute myeloid leukemiaThe caspase-8 inhibitor emricasan combines with the SMAC mimetic birinapant to induce

David L. Vaux, Paul G. Ekert and John SilkeGugasyan, Mark A. Gurthridge, Stephen M. Condon, Ricky W. Johnstone, Richard Lock, Guy Salvesen, Andrew Wei,Margherita Ghisi, Jessica M. Salmon, Natasha Silke, Giovanna Pomilio, Stefan P. Glaser, Elisha de Valle, Raffi Gabriela Brumatti, Chunyan Ma, Najoua Lalaoui, Nhu-Y Nguyen, Mario Navarro, Maria C. Tanzer, Jennifer Richmond,

DOI: 10.1126/scitranslmed.aad3099, 339ra69339ra69.8Sci Transl Med

for these patients.patients most likely to benefit from treatment with SMAC mimetics and selecting effective treatment combinationsthe apoptotic pathway and promotes cell death by necroptosis. These findings should be helpful for identifying

show that birinapant is particularly effective when combined with a caspase inhibitor, which shuts offet al.Brumatti when it can activate two different types of cell death: apoptosis and necroptosis. For acute myelocytic leukemia,

show that a SMAC mimetic called birinapant works bestet al.leukemia. For acute lymphocytic leukemia, McComb mimic the action of SMAC. Now, a pair of related articles provides insights into the effects of SMAC mimetics inmechanism of cell death that is commonly targeted by cancer therapies. SMAC mimetics are drugs designed to

Second mitochondria-derived activator of caspases, or SMAC, is a protein involved in apoptosis, aGiving leukemia a SMAC

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REFERENCES

http://stm.sciencemag.org/content/8/339/339ra69#BIBLThis article cites 44 articles, 17 of which you can access for free

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