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

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Science Translational Medicine  18 May 2016:
Vol. 8, Issue 339, pp. 339ra69
DOI: 10.1126/scitranslmed.aad3099
  • Fig. 1. AML driven by different oncogenes shows varying sensitivity to birinapant.

    (A) Schematic 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 terminal repeat; IRES, internal ribosomal entry site; E14, embryonic day 14; rmIL-3, recombinant murine interleukin-3; i.v., intravenously. (B) Survival of different AML cells in response to birinapant. Primary leukemic cells from four AML models were treated with birinapant for 16 hours. Cell survival data represent means ± SEM of three to six independent tumors per model. PI, propidium iodide. (C) Protein 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 treatment 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 treated with 100 nM birinapant ± TNFR1 antibody (10 μg/ml) for 16 hours. Data are means ± SEM, n = 3 independent tumors.

  • Fig. 2. Caspase inhibition sensitizes leukemic cells to birinapant.

    (A) Cells from primary MLL-ENL, MLL-AF9, and HoxA9 + Meis1 leukemias were pretreated for 15 to 30 min with the caspase inhibitors Z-VAD-FMK (Z-VAD) (5 μM) or Q-VD-OPh (Q-VD) (10 μM) followed by 100 nM birinapant treatment for 16 hours, after which cell viability was determined. Data are means ± SEM, n = 6. DMSO, dimethyl sulfoxide. (B) Schematic of TNF death signaling pathways associated with apoptosis and necroptosis. C8, caspase-8. (C) Kaplan-Meier survival curve of mice transplanted with MLL-ENL–transduced E14 liver cells of the indicated genotype. (D) MLL-ENL cells from wild-type (WT), Tnfr1−/−, Ripk3−/−, and Mlkl−/− mice were pretreated (15 to 30 min) with the caspase inhibitors Z-VAD-FMK (5 μM) or Q-VD-OPh (10 μM) followed by birinapant (100 nM). Cell viability was determined 16 hours after treatment. Means + SEM, n = 6. P values were obtained by comparing the survival of mice with WT and knockout leukemias treated with bir/ZVAD. (E) Birinapant-resistant MLL-ENL leukemic cells are sensitive to cell death induced by birinapant plus caspase inhibitors. Cells were pretreated (15 to 30 min) with the caspase inhibitors Z-VAD-FMK (10 μM) or Q-VD-OPh (10 μM) followed by 100 or 500 nM birinapant. Cell viability was determined 16 hours after treatment. Data are means ± SEM of two independent leukemias, each tested in two independent experiments (n = 4). (F) Human AML cell line MV4-11 was treated with birinapant ± Z-VAD-FMK (10 μM) or Q-VD-OPh (10 μM). Data are means ± SEM, n = 3. Cell survival was determined by propidium iodide uptake and flow cytometry. *P < 0.005, ***P < 0.0005, ****P ≤ 0.00005.

  • Fig. 3. Caspase-8 inhibitors are potent inducers of TNF-RIPK1–dependent cell death in AML cells.

    (A) MV4-11 cells were pretreated (15 to 30 min) or not with the caspase inhibitor IDN-6556 (5 μM) followed by birinapant at the specified doses for 48 hours. Data are mean ± SEM, n = 5. (B) MLL-ENL birinapant-sensitive (S) and MLL-ENL birinapant-resistant (R) leukemic cells were pretreated (15 to 30 min) with IDN-6556 (5 μM) or vehicle followed by birinapant (100 nM) or vehicle. Cell viability was 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 inhibitors IDN-6556 (5 μM) or Q-VD-OPH (10 μM) followed by birinapant (100 nM for MLL-AF9 cells and 500 nM for HoxA9 + Meis1 and Nup98-HoxA9) ± Nec-1 (50 μM) for 16 hours. (D to F) TNF concentration in total cell lysates of MLL-ENL birinapant-sensitive and birinapant-resistant, MLL-AF9, and HoxA9 + Meis1 cells was determined after birinapant (100 nM for MLL-ENL and MLL-AF9 and 500 nM for HoxA9 + Meis1), IDN-6556 (5 μM), or bir/IDN treatments for the indicated time period. Data are means ± SEM (n = 4 to 9) of three independent experiments. (G to I) TNF concentrations in total cell lysates of MLL-ENL birinapant-sensitive (S) and birinapant-resistant (R), MLL-AF9, and HoxA9 + Meis1 cells were determined after 9 hours of treatment with bir/IDN ± Nec-1 (50 μM). Drug concentrations were the same as in (D) to (F). Data are means ± SEM, n = 3 to 4. P value was determined by comparison of bir/IDN and bir/IDN + Nec-1 for each leukemia subtype. Cell survival was determined by propidium iodide uptake and flow cytometry. *P < 0.05, **P < 0.005, ***P < 0.0005.

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

    (A) MLL-ENL leukemias derived from WT, Tnfr1−/−, Ripk3−/−, Mlkl−/−, Casp8−/−Ripk3−/−, and Casp8−/−Mlkl−/− progenitors were pretreated with IDN-6556 (5 μM) or vehicle followed by birinapant (50 nM) or vehicle. Graphs: Cell viability was determined 16 hours after treatment with propidium iodide staining and flow cytometry. Mean ± SEM, n = 6. Images: Clonogenic assay to determine long-term survival and proliferation of MLL-ENL leukemia cells treated with birinapant (250 nM) ± IDN-6556 (5 μM). Cells were pretreated for 30 min with IDN-6556 followed by birinapant. After the pretreatment, 500 cells were plated in 0.3% agar containing birinapant or bir/IDN. Clonogenic survival was determined 15 days after treatment. (B) TNF production in WT, Tnfr1−/−, Ripk3−/−, and Mlkl−/− MLL-ENL leukemias. Cells were treated with birinapant (100 nM) ± IDN-6556 (5 μM) for 9 hours, and TNF concentrations were determined in whole-cell lysates by enzyme-linked immunosorbent assay (ELISA). Mean ± SEM, n = 5. P values were obtained by comparison of birinapant- and bir/IDN-treated leukemic cells from each genotype. (C) Time course of killing of MLL-ENL leukemias derived from WT, Mlkl−/−, and Ripk3−/− progenitors pretreated with IDN-6556 (5 μM) followed by birinapant (100 nM). P values were calculated by comparing WT leukemic cells with the specific knockout at each time point: 8, 10, and 16 hours. (D) MLL-ENL leukemias derived from WT, Mlkl−/−, and Ripk3−/− progenitors were pretreated with IDN-6556 (5 μM) followed by birinapant (100 nM) ± Q-VD-OPH (10 μM). (E) MLL-ENL leukemias derived from WT, Mlkl−/−, and Ripk3−/− progenitors were pretreated with IDN-6556 (5 μM) followed by birinapant (100 nM). IDN-6556 was readded to cells 3 hours after birinapant treatment, and cell survival was determined 16 to 18 hours later. Data are means ± SEM, n = 4. (F) MLL-ENL Casp8−/−Mlkl−/− leukemia cells were transduced with a lentiviral construct expressing MLKL from a doxycycline (dox)–inducible promoter. Leukemic cells were treated with birinapant ± IDN-6556 in the presence or absence of doxycycline (0.5 μg/ml) to induce MLKL. Cell viability was determined 16 hours after treatment. Mean ± SEM, n = 4. (G) Comparison of the ability of Z-VAD-FMK, Q-VD-OPH, and IDN-6556 to inhibit recombinant caspase-8 homodimer and caspase-8/cFLIP heterodimer. Values are means ± SD of duplicate experiments. Cell survival was determined by propidium iodide uptake and flow cytometry. *P < 0.05, **P < 0.005, ***P < 0.0005.

  • Fig. 5. Combination of birinapant and the clinical caspase-8 inhibitor emricasan/IDN-6556 prolongs survival in AML models.

    (A) In vivo imaging of tumor progression in mice treated with vehicle (6% Captisol in phosphate-buffered saline), IDN-6556 (2.5 mg/kg), birinapant (5 mg/kg), or the combination. Mice were treated with the drugs twice a week for 4 weeks, starting treatment 10 to 11 days after retransplant of tumor cells. Images are representative of animals from each treatment group at the specified time point. i.p., intraperitoneal. (B) Graphs represent average photons per second values from mice treated with birinapant, IDN-6556, or the combination. Error bars represent SEM of n = 14 for vehicle, n = 10 for IDN-6556, n = 14 for birinapant, and n = 16 for bir/IDN. (C) Kaplan-Meier survival curve of albino C57BL/6 mice transplanted with MLL-ENL leukemia and treated with vehicle, birinapant, IDN-6556, or the combination (bir/IDN). Arrows indicate the start and end of treatment schedule. Drug concentrations used as described in (A). Statistical analysis was performed by comparison of birinapant or bir/IDN-treated mice with vehicle (blue) or IDN-6556 (orange). n.s., not significant. (D) Hematoxylin and eosin–stained sections of spleen and liver from mice with MLL-ENL AML after treatment with IDN-6556, birinapant, or the combined therapy. Arrows indicate infiltration of blast leukemic cells. Scale bars, 200 μm for the (spleen images) and 100 μm (liver images). (E) bir/IDN treatment is effective in ALL (acute lymphoblastic leukemia) and AML PDX samples. Median inhibitory concentration (IC50) of birinapant or bir/IDN treatment was determined for these PDX samples. Cells were treated for 48 hours with birinapant (10 to 1000 nM) ± IDN-6556 (5 μM), and cell viability was determined by annexin V/7-AAD staining. Data are means of four independent experiments. N/A, not applicable. (F) Combined therapy kills patient leukemia samples. Primary leukemic cells derived from patients with the indicated treatment status were treated for 24 hours with birinapant (500 nM) ± IDN-6556 (5 μM). Cell viability was determined by flow cytometry of CD34+ propidium iodide–negative cells. Data are means of two independent experiments. *P < 0.05, **P < 0.005. hCD34, human CD34.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/339/339ra69/DC1

    Fig. 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 or RIPK1 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.

    Fig. S8. Comparison of the ability of caspase inhibitors to induce or block cell death in leukemic 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.

  • Supplementary Material for:

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

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

    *Corresponding author. Email: silke{at}wehi.edu.au (J.S.); paul.ekert{at}mcri.edu.au (P.G.E.)

    Published 18 May 2016, Sci. Transl. Med. 8, 339ra69 (2016)
    DOI: 10.1126/scitranslmed.aad3099

    This PDF file includes:

    • Fig. 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 or RIPK1 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.
    • Fig. S8. Comparison of the ability of caspase inhibitors to induce or block cell death in leukemic 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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