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1 Supplementary Materials 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. (J.S.); (P.G.E.) The PDF file includes: Published 18 May 2016, Sci. Transl. Med. 8, 339ra69 (2016) DOI: /scitranslmed.aad3099 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.

2 Supplementary Materials: Fig. S1: WB: α-ciap1 MLL-AF9 MLL-ENL HOXA9+Meis1 Nup98-HOXA9 (kd) 70 WB: α-xiap exposure s 50 α-xiap exposure L 50 WB: α-tnfr1 50 WB: α-casp 8 50 WB: α-ripk1 80 WB: α-ripk3 50 WB: α-mlkl 50 WB: α-p65 60 WB: α-actin 40 Fig. S1. Endogenous protein expression in the different AML subtypes. The expression of the indicated proteins was determined in total cell lysates of 2 to 3 independent leukemias from MLL-ENL, MLL-AF9, HoxA9+Meis1, and NUP98-HoxA9 models by Western blotting and probing with the indicated antibodies. Actin was used as a loading control.

3 Fig. S2:

4 Fig. S2. Generation of single-gene knockout leukemias. (A-B) All mice transplanted with MLL-ENL transformed cells presented with enlarged spleen (splenomegaly) and high white blood cell count (WBC) as indicators of AML development. (C-D) Genotype of MLL-ENL leukemias generated from knock-out mice was confirmed by protein and PCR analysis. (C) Western blots of lysates from 3 different AMLs of the indicated genotypes were probed with the indicated antibodies. Actin was used as a loading control. (D) AML and control samples were genotyped for Tnfr1 using a standard PCR-based assay that detects the knock-out and wild type alleles. (E) MLL-ENL leukemic cells derived from WT and Tnfr1 -/- cells were treated with indicated concentrations of birinapant for 16 hours. Mean ± SEM, n=3 independent tumors per genotype. Cell survival was determined by PI uptake and flow cytometry.

5 Fig. S3.

6 Fig. S3. Generation of MLL-ENL birinapant-resistant cells. (A) Schematic of generation of MLL-ENL birinapant-resistant cells. MLL-ENL sensitive cells were treated for 4 weeks with increasing concentrations of birinapant followed by sorting of surviving cells (PI-negative) after 16 hours of treatment with 2 μm birinapant. (B) MLL-ENL birinapant-treated PI-negative cells were tested for resistance to birinapant. Cells were cultured with 1000 nm birinapant or μm ara-c for 18 hours. Cell survival was determined by flow cytometry and PI staining. Data represent mean ± SEM of n=4 from 2 independent leukemias. (C) Comparison of protein expression in MLL-ENL sensitive and resistant leukemias after 8 hours of treatment with birinapant (500 nm). (D) Cell survival of MLL-ENL sensitive (S) and resistant (R) leukemias after treatment with birinapant (100 nm) or birinapant+idn-6556 (5 μm) for the indicated time period. Data represent mean ± SEM of n=3 from 3 independent tumors. (E) MLL-ENL sensitive (S) and resistant (R) leukemias were pre-treated for 30 min with IDN (5 μm) or Q-VD (10 μm) followed by birinapant (100 nm) ± Nec-1 (50 μm). Cell survival was determined after hours of treatment. Data represent mean ± SEM n=6. (F) Western blot analysis of birinapant sensitive (S) and resistant (R) MLL-ENL cells treated for 5 hours with birinapant (500 nm), IDN-6556 (5 μm), or the combination and probed with the indicated antibodies. Actin expression serves as loading control. Results are representative of 2 repeats.

7 Fig. S4.

8 Fig. S4. IDN-6556 mediated TNF-dependent necroptosis in different AML subtypes. (A) MLL-ENL wild-type cells or those with acquired resistant to ara-c ( ara-c R ) were treated with 100 nm birinapant ± IDN-6556 (5 μm) or increasing concentrations of ara-c (100, 500, and 1000 nm). Cell viability was determined by PI staining 16 hours after treatment. Data represent mean ± SEM of n=5. (B) MLL-AF9, MLL-ENL, AML-ETO9a, and CBFß/MYH11 constitutively expressing activated NRAS were pre-treated for 30 min with IDN (5 μm) or vehicle followed by birinapant (500 nm) or vehicle. Cell survival was determined after 24 hours of treatment. Data represent mean ± SEM of n=3. (C) Bir/IDN resistant cells, AML- ETO9a and CBFß/MyH11, were treated with bir (1 μm) or bir+idn (10 μm) ± 100 ng/ml of recombinant TNF. Cell death was determined by PI exclusion at 24 hours after treatment. Data represent mean ± SD of n=2 from 2 independent experiments. (D) Comparison of protein expression in the bir/idn sensitive (MLL-ENL, MLL-AF9, Nup98HoxA9, HoxA9+Meis1) and resistant (AML-ETO9a and CBFß/MYH11) leukemias. Total cell lysates were analyzed for the indicated proteins. Actin was used as loading control. Western blot shown is representative of 3 repeats. (E) Bax -/- Bak -/- hematopoietic stem cells transduced with MLL- ENL were pre-treated or not with IDN (5 μm) followed by the addition of birinapant (100 nm) or vehicle. Cell survival was determined 16 hours after treatment. Data represent mean ± SEM of n=3.

9 Fig. S5. Fig. S5. Prevention of IDN-6556 induced cell death in human AML cells by inhibiting TNFR1 or RIPK1 kinase activity. (A-B) Survival of human leukemic cells, MV4-11 and U937, 24 and 48 hours after birinapant (500 nm) ± IDN (5 μm) treatment ± Nec-1 (50 μm). (C-D) Survival of human leukemic cells, MV4-11 and U937, 24 hours after birinapant (500 nm) ± IDN (5 μm) treatment ± TNFR1 blocking antibody (100 ng/ml). All cell death was determined by PI exclusion. Data represent mean ± SM of n=3.

10 Fig. S6.

11 Fig. S6. Generation of MLL-ENL double-gene knockout leukemias. (A) Kaplan-Meier survival plot for MLL-ENL leukemias generated from wild type, Casp8 -/- Ripk3 -/-, and Casp8 -/- Mlkl -/- hematopoietic cells, n=3 from each genotype. (B) Spleen and liver weight and white blood cell count from mice reconstituted with MLL-ENL WT, Casp8 -/- Ripk3 -/-, and Casp8 -/- Mlkl -/- leukemic cells. (C) Expression of caspase-8, RIPK3, and MLKL in 3 biologically independent MLL-ENL leukemias derived from the indicated strains of mice. (D) Representative Western blot of MLKL, RIPK3, cleaved caspase-8 and -3 in WT, Mlkl -/-, and Ripk3 -/- MLL-ENL cells after bir (100 nm)+idn (5 μm) treatment for the indicated period of time. (E) Re-expression of MLKL in Casp8 -/- Mlkl -/- MLL-ENL leukemic cells. Cells were treated vehicle or 0.5 μg/ml doxycycline for 16 hours. Western blots of lysates were probed with antibody against MLKL. Actin was used as a loading control.

12 Fig. S7.

13 Fig. S7. Biochemical comparison of caspase inhibitors IDN-6556, Z-VAD-FMK, and Q- VD-OPh. The graphs show Kobs/[I] for caspase inhibitors Q-VD, Z-VAD, and IDN-6556 vs caspase-8 homodimers, caspase-8/cflip heterodimers, caspase-3, and caspase-9.

14 Fig. S8.

15 Fig. S8. Comparison of the ability of caspase inhibitors to induce or block cell death in leukemic cells. (A) MLL-ENL leukemic cells were pre-treated (30 min) with the indicated concentrations of Q-VD, Z-VAD, or IDN-6556 followed by birinapant (100 nm) or vehicle. Cell survival was assessed by PI staining and flow cytometry after 16 hours. Data represent mean ± SEM, n=6. (B) Protein expression in MLL-ENL leukemic cells pre-treated with 10 μm of each caspase inhibitor Q-VD, Z-VAD, or IDN-6656, followed by 100 nm of birinapant. Total cell lysates were prepared 5 hours after treatment and probed with the indicated antibodies. Representative data of 3 independent leukemias. (C) Comparison of the ability of the caspase inhibitors IDN-6556 and Q-VD to inhibit ara-c-mediated apoptotic cell death. MLL-ENL leukemic cells were pre-treated (30 min) with 10 μm of Q-VD or IDN-6556 followed by the indicated doses of ara-c. Cell survival was determined by PI staining and flow cytometry 16 hours after treatment. Data represent mean ± SEM, n=3.

16 Fig. S9. Fig. S9. Combined treatment with bir/idn in vivo. (A) No toxicity observed in vivo for combined birinapant plus IDN-6556 therapy. Measurements of body weight, spleen, and liver

17 from C57BL/6 female mice treated for 4 weeks (i.p. injections 2x/week) with birinapant (7.5 mg/kg or 10 mg/kg) + IDN-6556 (2.5 mg/kg). Organs and blood were analyzed 4-6 weeks after treatment stopped. (B-C) Kaplan-Meier survival curves of C57BL/6 mice transplanted with MLL-ENL birinapant-resistant or MLL-AF9 leukemic cells treated (i.p.) with the indicated drugs. Drugs were administered i.p. starting 3 days after retransplant for a period of 4 weeks with 2 injections a week. Mice were analyzed when signs of disease (anemia, enlarge spleen, hunched posture) were observed. P values were obtained by comparing mice from bir or bir+idn treated groups to vehicle.

18 Fig. S10.

19 Fig. S10. Safety of combined bir/idn treatment in healthy human cells. (A) CD34+ stem cells from 3 independent control patients, were treated for 48 hours with birinapant (0, 250, or 500 nm) ± IDN-6556 (5 µm) or conventional chemotherapies: cytarabine (10 µm) or daunorubicin (0.4 µm). Cell viability was determined by flow cytometry of PI-negative cells. Data represent means of 3 independent experiments. (B) Colony forming assays were performed on CD34+ mobilized peripheral blood progenitor cells from 2 independent patients. Birinapant and IDN were added to plates on day 0 at the indicated concentrations. Colony numbers were normalized to DMSO control after 14 days of incubation. (C) PBMC, CD3+CD4+ T cells, CD19+ B cells, and CD16+ NK cells from 3 independent control patients were treated for 48 hours with birinapant (1000 nm) ± IDN-6556 (5 µm) or conventional chemotherapies: cytarabine (10 µm), daunorubicin (0.4 µm), or idarubicin (0.4 µm). Cell viability was determined by flow cytometry of PI-negative cells. Data represent means of 3 independent experiments.

20 Patient Age Sex WHO classification 1 60 M AML with myelodysplastic related changes (progression from MDS) 2 44 M AML relapsing post-allograft Blasts Karyotype MLL WBC Other (x10 9 /L) 11q % del(7) Neg - (p13p15) % Normal Neg FLT3-ITD IDH1 R132C 3 28 F Acute myelomonocytic leukemia % Normal Neg FLT3-ITD DNMT3A R882H 4 57 F Acute myelomonocytic leukemia % Normal Neg FLT3-ITD NPM M AML with inv (3) % inv(3) (q21q26) Neg FLT3-ITD 6 51 M AML with inv (16) % inv(16) (p11.2q22) Neg - 7?? AML with MLL??? Pos M AML with inv (16) % inv(16) (p13q22) Neg FLT3-ITD Table S1 - Deidentified patient data.