CT-2A * * * * * * NRRP (particles per cell)

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1 CT-2A Viability (%) TNF-α: AT-46 GDC-917 LCL161 Monomer AZD-5582 Birinapant Dimer Supplementary Figure 1. s potently synergize with TNF-α to induce the death of glioblastoma cells. Viability of mouse glioblastoma CT-2A cells to the treatment of.1% BSA or.1 ng ml -1 TNF-α and vehicle or 5 μm of the indicated monomeric or dimeric for h. Viability was assessed by Alamar blue. Error bars, mean, s.d. n = 4. Representative data from two independent experiments using biological replicates. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett s multiple comparison test. Significance is reported if p <.1 (). Viable cells (%) NRRP (particles per cell) CT-2A DBT Supplementary Figure 2. s synergize with non-replicating rhabdovirus-derived particles (NRRPs) to induce the death of glioblastoma cells. Alamar blue viability assays of mouse glioblastoma CT-2A or DBT-cells to the treatment of vehicle or 5 μm LCL161 () and increasing numbers of NRRPs for h. Error bars, mean, s.d. n = 3. Representative data from two independent experiments using biological replicates.

2 a Viable cells (%) K51 NT sirna: cflip sirna: BSA TNF-α VSV 51 G (MOI) SF539 Viable cells (%) NT sirna: cflip sirna: BSA TNF-α VSV 51 G (MOI) 12 GM38 Viable cells (%) NT sirna: cflip sirna: BSA TNF-α VSV 51 G (MOI) b SF539 NT sirna: cflip sirna: cflip β-tubulin K51 GM Supplementary Figure 3. Resistance to -based combinations in glioblastoma cells is circumvented with downregulation of cflip. (a) Primary mouse NF1-/p53-/ (K51) or human (SF539) glioblastoma cells or human non-transformed cells (GM38) were transfected with nontargeting (NT) or cflip sirna for h and subsequently treated for h with vehicle or 5 μm LCL161 () and BSA,.1 ng ml -1 TNF-α or the indicated MOI of a nonspreading version of VSVΔ51 (VSVΔ51ΔG). Viability was determined by Alamar blue. Error bars, mean, s.d. n = 4. Representative data from three independent experiments using biological replicates. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett s multiple comparison test. Significance is reported if p <.1 (). (b) Efficacy of NT sirna or sirna targeting cflip from the experiment in (a).

3 a PBS CT-2A b Coronal Horizontal CT-2A PBS Supplementary Figure 4. Establishment of a mouse syngeneic orthotopic model of glioblastoma. MRI (a) and gross (b) images of a C57BL/6 mouse injected intracranially with PBS or 5x14 CT-2A cells and sacrificed at days post-implantation. Scale bar, 2 mm. Ruler is in cm with mm divisions. Cortex Cerebellum Spleen (h): ciap1/2 XIAP β-tubulin ciap1/2 XIAP β-tubulin ciap1/2 XIAP β-tubulin Supplementary Figure 5. treatment does not induce the downregulation of the IAPs in brain tissue from non-tumor bearing mice. Mice were treated with mg kg -1 of LCL161 () for the indicated time, and tissues were processed for Western Blotting using the indicated antibodies. n = 2 for each timepoint. (h): ciap1/2 XIAP β-tubulin CT-2A brain tumours Supplementary Figure 6. Degradation of ciap1/2 and XIAP in intracranial gliomas upon intratumoral injection of a. Mice with 21 d intracranial CT-2A tumors were treated by stereotactic injection (at the same coordinates as with the initial tumor implantation) with 1 μl of μm LCL161 (), and tumors were processed at the indicated times post-treatment for Westerm blotting using the indicated antibodies. n = 2 for control and n = 3 for -treated mice at the indicated post-treatment time.

4 Survival probability poly(i:c) treatment in C57BL/6 mice n = Time (days post-treatment) Supplementary Figure 7. Intracranial treatment of poly(i:c) does not cause mortality. Kaplan-Meier curve depicting survival of mice that received three 1 μl intracranial treatments of 5 μg poly(i:c). Treatments were on days, 3, and 7. Normalized counts SNB PD-L1 (BV421) PD-L2 (AF647) HLA-A,B,C (PE) HLA-DR/DP/DQ (BV65) Isotype control BSA TNF-α IFN-β VSV 51 Supplementary Figure 8. treatment does not abrogate expression of checkpoint inhibitor molecules or MHC I/II proteins. SNB cells were treated for 24 h with vehicle or 5 μm LCL161 () and 1 ng ml -1 TNF-α, 25 U ml -1 IFN-β or.1 MOI of VSVΔ51, and viable cells (Zombie Green negative) were processed for flow cytometry using the indicated antibodies. Representative data from three independent experiments.

5 Spots per 1, cells IFN-γ CD8 T-cell Naïve: Cured: Cancer cell EMT6: line 4T1: α-pd-1: GrzB Supplementary Figure 9. CD8 T-cells cells from mice previously cured of mammary tumors recognize the same tumor cell type. CD8 T-cells were enriched from naïve mice or mice previously cured of EMT6 tumors, and subjected to ELISpot assays for the detection of IFN-γ and GrzB. Cancer cells (EMT6, 4T1) were cocultured with CD8 T-cells (12:1 ratio) and 1 μg ml -1 of control IgG or α-pd-1 for h. Error bars, mean, s.d. n = 3 of mice for naïve or cured groups. Significance was compared to naïve CD8 T-cell co-incubated with EMT6 cells as assessed by ANOVA with Dunnett s multiple comparison test. p <.1;, p <.1. FSC 25K 2K 15K K CD45 (BV65) Counts CD45 CD K CD3 (APC-Cy7) CD3 (APC-Cy7) CD8 (PE) CD8 (PE) Normalized counts CD45 CD3 CD45 CD3 CD PD-1 (BV421) PD-1 (BV421) Normalized counts PD-1 (BV421) PD-1 (BV421) Supplementary Figure 1. treatment leads to the upregulation of PD-1 in CD8 T-cells. Mice bearing intracranial CT-2A tumors were treated with mg kg -1 LCL161 orally () on post-implantation days 14, 16, 21 and 23. Viable cells from CT-2A tumors were processed for flow cytometry using the antibodies CD45 (BV65), CD3 (APC-Cy7), CD8 (PE) and PD-1 (BV421). Data represented as box and whisker plots are in Fig. 4b.

6 a Viable cells (%) TNF-α: IFN-α: IFN-β: MPC-11 b Normalized To Mode MPC PE-Cy7 IgG α-pd-l1 c α-pd-1 (i.p.): (p.o.): Survival probability MPC Time (d post-implantation) (4) (7) (4) (4) (4) (4) IgG α-pd-1 α-ctla-4 d IgG α-ctla-4 α-pd-1 3. Time (d post-implantation) x1 6 Supplementary Figure 11. s synergize with immune checkpoint inhibitors for the treatment of a mouse model of multiple myeloma. (a) MPC-11 cells were treated with vehicle or 5 μm LCL161 () and.1 ng ml -1 TNF-α, 25 U ml -1 IFN-α or 25 U ml -1 IFN-β. Viability was determined by Alamar blue at h post-treatment. Error bars, mean, s.d. n = 4. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett s multiple comparison test. Significance is reported if p <.1 (). Representative data from three independent experiments using biological replicates. (b) MPC-11 cells were dissociated and processed for flow cytometry with PE-Cy7-conjugated isotype IgG or PD-L1. Representative data from two independent experiments. (c) Mice were injected with murine MPC-11 cells intravenously, and 5 days later were treated at the indicated time points with combinations of vehicle or mg kg -1 LCL161 orally and 25 μg of IgG, α-pd-1 or α-ctla4 intraperitoneally. Data represents the Kaplan-Meier curve depicting mouse survival. Log-rank with Holm-Sidak multiple comparison:, p <.5. Numbers in parentheses represent number of mice per group. Representative data from two independent experiments. (d) Representative IVIS images from mice described in (c) were acquired at indicated time points. Scale: p/sec/cm2/sr.

7 a Viable cells (%) TNF-α: IFN-β: VSV 51: EMT6 b Normalized counts 1 EMT PE-Cy7 IgG α-pd-l1 c VSV 51 (i.t.): α-pd-1 (i.p.): (p.o.): Tumour volume (mm 3 ) 2,5 2, 1,5 1, 5 EMT6 mammary tumors Time (d post-treatment) Survival probability EMT6 mammary tumors Time (d post-treatment) (4) (5) (4) (5) PBS VSV 51 α-pd1 Supplementary Figure 12. The combination of s with antibodies targeting immune checkpoint inhibitors in a mouse model of mammary cancer. (a) Viability assay of EMT6 cells treated with vehicle or 5 μm LCL161 () and.1 ng ml -1 TNF-α, 25 U ml -1 IFN-β or.1 MOI of VSVΔ51 for h. Error bars, mean, s.d. n = 4. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett s multiple comparison test. Significance is reported if p <.1 (). Representative data from three independent experiments using biological replicates. (b) EMT6 cells were dissociated and processed for flow cytometry with PE-Cy7-conjugated isotype IgG or PD-L1. Representative data from three independent experiments. (c) Mice bearing ~ mm 3 EMT6-Fluc tumors were treated at the indicated post-implantation times with PBS or 5x1 8 PFU of VSVΔ51 intratumorally, and then with vehicle or 5 mg/kg LCL161 () orally and 25 μg of IgG or α-pd-1 intraperitoneally. Left panel depicts tumor growth. Error bars, mean, s.e,m. Right panel represents the Kaplan-Meier curve depicting mouse survival. Log-rank with Holm-Sidak multiple comparison:, p <.5;, p <.1. Numbers in parentheses represent number of mice per group.

8 a TNF-α (pg ml -1 ) IL-6 (pg ml -1 ) IL-17 (pg ml -1 ) IFN-γ (pg ml GrzB (ng ml -1 ) -1 ) CT-2A: Naïve: Splenocytes Cured: mab α-pd-1: α-pd-l1: b IFN-γ (pg ml -1 ) IL-6 (pg ml -1 ) IL-17 (pg ml -1 ) 3, 2,5 2, 1,5 1, 5 4 GrzB (ng ml -1 ) TNF-α (pg ml -1 ) IL-1 (pg ml -1 ) Supplementary Figure 13. The inclusion of s increases the immune response in the presence of glioblastoma cells. (a) The expression of the indicated factors was detected by ELISA from cell culture supernatants of CT-2A cells that were co-incubated for h with splenocytes derived from naïve mice or mice previously cured with intracranial CT-2A tumors by and anti-pd-1 cotreatment (1:2 ratio of CT-2A cells to splenocytes). Data represented as heat maps is in Fig. 5a. Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to th percentile, and whiskers depicts min-max range of the values. Statistical significance was compared to naïve CD8 T-cell as assessed by ANOVA with Dunnett s multiple comparison test., p <.5; p <.1;, p <.1. (b) The indicated cytokines were determined by ELISA from CT-2A cells that were cocultured with splenocytes derived from naïve or cured mice and treated with vehicle or 5 μm LCL161 () for h. Heat map plots are shown in Fig. 5a.. Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to th percentile, and whiskers depicts min-max range of the values. Statistical significance was compared to vehicle and IgG treated T-cells as assessed by ANOVA with Dunnett s multiple comparison test. p <.1;, p <.1. CT-2A: : α-pd-1:

9 pg ml -1 5 IFN-β 15 5 IFN-γ 1 5 IL-1α IL-1β IL-6 IL-1 IL-12p7 IL-17A : α-pd-1: pg ml -1 5 IL IL GM-CSF MCP TNF-α : α-pd-1: Supplementary Figure 14. Combinatorial and immune checkpoint inhibitor treatment leads to the increased systemic presence of proinflammatory cytokines. Serum from mice depicted in Fig. 6a was processed for multiplex ELISA for the quantitation of the indicated proteins. Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to th percentile, and whiskers depicts min-max range of the values. Significance was compared to vehicle and IgG treated mice as assessed by ANOVA with Dunnett s multiple comparison test., p <.5. n = 6 for each treatment group. Data represented as heat maps is in Fig. 7e.

10 : α-pd-1: Min Tnfsf11 Inhba Tgfb1 Tgfb3 Bmp1 Ccl11 Il17c Il1f8 Tnfsf9 Inhbe Cd4l Angpt4 Tgfb2 Bmp8a Amh Ntf5 Il17b Nodal Gdf5 Gdf11 Inhbc Gdf1 Il1f6 Bmp8b Cd7 Ccl21a Ccl8 Ccl28 Eda Lefty2 Fgf16 Tnfsf12 Lta Tnfsf8 Ltb Tnfsf15 Gdf3 Tnfsf1 Ccl17 Il1a Bmp5 Ccl19 Tnfsf13b Ccl3 Cxcl16 Cxcl2 Cxcl3 Gdf15 Tnfsf14 Il24 Fgf2 Il23a Egf Il1b Tnfsf4 Cxcl1 Cxcl11 Ccl24 Ccl2 Ccl4 Ccl5 Fasl Gdnf Ccl22 Tnf Cxcl9 Cxcl13 Il2 Ccl7 Il1rn Il19 Ifnb1 Ccl25 Fgf15 Fgf22 Bmp7 Bmp4 Gdf1 Il17d Mstn Il18 Inhbb Inha Bmp2 Gdf7 Efnb2 Cxcl5 Cxcl14 Bmp6 Fgf9 Ntf3 Fgf13 Fgf5 Nrg3 Fgf14 Bdnf Nrg4 Gas6 Ccl2 Il1f5 Bmp3 Ccl27 Fgf1 Cxcl1 Bmp15 Fgf21 Ppbp Pdgfd Epo Il12a Fgf12 Prl Fgf17 Csf3 Pdgfc Fgf11 Nrg1 Gdf9 Il11 Il1 Tnfsf18 Bmp1 Il4 Osm Efna4 Il17f Ifne1 Fgf7 Efnb1 Vegfc Ngfb Il9 Il13 Ifng Gh Il15 Clc Il12b Tslp Angpt2 Fgf4 Figf Il6 Hgf Angpt1 Cxcl12 Lefty1 Fgf18 Ctf1 Zfp91 Lep Il5 Csf1 Vegfa Igf1 Fgf6 Max Supplementary Figure 15. Proinflammatory cytokine and chemoattractant chemokine gene signatures are upregulated with and immune checkpoint inhibitor combinatorial treatment. Mice were treated as in Fig. 6a, and intracranial CT-2A tumors were processed for quantitation of 176 cytokine and chemokine genes by RT-qPCR. Shown are normalized heat maps of major groups identified by hierarchical clustering. n = 4 for each treatment group.

11 CD8 Proportion (%) CD8 CT-2A: : α-pd-1: CT-2A: : α-pd-1: 1 3 CFSE Supplementary Figure 16. s enhance clonal expansion of CD8 T-cells in the presence of glioblastoma target cells. Isolated splenic CD8 T-cells derived from mice previously cured of CT-2A tumors were loaded with CFSE and co-incubated with CT-2A cells (1:1 ratio) for 96 h in the presence of vehicle or 5 μm LCL161 () or 2 μg ml -1 of control IgG or anti-pd1. Viable cells were processed for flow cytometry. Significance was compared to vehicle and IgG treated mice as assessed by ANOVA with Dunnett s multiple comparison test., p <.5;, p <.1;, p <.1. n = 5 for each treatment group.

12 2 3 4 Polarization Sensitization to TNF-α Co-stimulation (e.g. CD4L) Macrophage (M1>>M2) Tumor TNFα Engulfment or immunogenic cell death 1 Cytokine and chemokine production (e.g. TNF-α) Immune cell recruitment T-cell 6 5 APC (DC) Tumor Co-stimulation antigen (e.g. GITR, CD27, presentation 4-1BB, OX4L) Reversal of immune checkpoint blockade (e.g. α-pd-1, α-pd-l1) CTL Supplementary Figure 17. s are immunoregulatory drugs that act on tumor and immune cells to eradicate cancer through the innate and adaptive immune systems. Shown is a model depicting the single agent and combinatorial immunomodulatory effects of Smac mimetics based on our results and previously published findings. The effects of IAP antagonism on these immune or tumor cells are outlined below: (1) s stimulates the production of cytokines and chemokines from various immune cells, such as macrophages or T-cells, which results in infiltration of immune cells within the tumor microenvironment 1-3. (2) treatment decreases the immunosuppressive macrophage M2 population and concomitantly increases the pro-inflammatory M1 population 4. (3) s deplete ciap1 and ciap2 to sensitize tumors to death by immune ligands, such as TNF-α or TRAIL 1,5-8. Tumor cell death is sensed by the immune system resulting in the priming of a cytotoxic T-cell (CTL) response. (4) s stimulate the TNF/TNFR family member CD4L/CD4 signaling pathway on antigen-presenting cells (APCs) to promote the differentiation and maturation of dendritic cells (DCs) and macrophages 9. APCs present tumor antigens to the immune system and further release cytotoxic inflammatory cytokines. (5) As a consequence of degrading ciap1 and ciap2 by treatment, s activate the alternative NF-κB pathway, removing the need for a TNF superfamily ligand (such as 4-1BB) and therefore providing a T-cell costimulatory signal 1, 11. (6) s have been shown to increase CTL and natural killer cell mediated cell death 12, 13. Granzyme B-mediated cell death is blocked by the X-linked IAP, XIAP, and this block can be overcome by the mitochondrial release of Smac or by its drug mimic,

13 a ciap1/2 ciap1/2 XIAP XIAP E7 E7 b cflip β-tubulin cflip β-tubulin cflip β-tubulin c ciap1/2 XIAP E ciap1/2 XIAP E ciap1/2 XIAP E d ciap1/2 XIAP E7 Supplementary Figure 18. Full-length blots pertaining to (a) Fig. 2a, (b) Supplementary Fig. 3b, (c) Supplementary Fig. 5 and (d) Supplementary Fig. 6.

14 Suppplementary References 1. Beug, S.T. et al. Smac mimetics and innate immune stimuli synergize to promote tumor death. Nat Biotechnol 32, (214). 2. Infante, J.R. et al. Phase I dose-escalation study of LCL161, an oral inhibitor of apoptosis proteins inhibitor, in patients with advanced solid tumors. J Clin Oncol 32, (214). 3. Erickson, R.I. et al. Toxicity profile of small-molecule IAP antagonist GDC-152 is linked to TNF-alpha pharmacology. Toxicological sciences : an official journal of the Society of Toxicology 131, (213). 4. Lecis, D. et al. Smac mimetics induce inflammation and necrotic tumour cell death by modulating macrophage activity. Cell death & disease 4, e92 (213). 5. Vince, J.E. et al. IAP antagonists target ciap1 to induce TNFalpha-dependent apoptosis. Cell 131, (27). 6. Gaither, A. et al. A Smac mimetic rescue screen reveals roles for inhibitor of apoptosis proteins in tumor necrosis factor-alpha signaling. Cancer Res 67, (27). 7. Li, L. et al. A small molecule Smac mimic potentiates TRAIL- and TNFalpha-mediated cell death. Science, (24). 8. Fulda, S., Wick, W., Weller, M. & Debatin, K.M. Smac agonists sensitize for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo. Nat Med 8, (22). 9. Muller-Sienerth, N. et al. SMAC mimetic BV6 induces cell death in monocytes and maturation of monocyte-derived dendritic cells. PLoS One 6, e21556 (21). 1. Zarnegar, B.J. et al. Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors ciap1, ciap2, TRAF2 and TRAF3 and the kinase NIK. Nature Immunology 9, (28). 11. Dougan, M. et al. IAP inhibitors enhance co-stimulation to promote tumor immunity. The Journal of experimental medicine 27, (21). 12. Kashkar, H. et al. XIAP targeting sensitizes Hodgkins Lymphoma cells for cytolytic T cell attack. Blood (26). 13. Brinkmann, K. et al. Second mitochondria-derived activator of caspase (SMAC) mimetic potentiates tumor susceptibility toward natural killer cell-mediated killing. Leukemia & lymphoma 55, (214). 14. Goping, I.S. et al. Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition. Immunity 18, (23). 15.Ravi, R. et al. Resistance of cancers to immunologic cytotoxicity and adoptive immunotherapy via X-linked inhibitor of apoptosis protein expression and coexisting defects in mitochondrial death signaling. Cancer Res 66, (26).