Supplementary Figure 1 Supplementary Figure 1. TWEAK is internalized and degraded following binding to Fn14-positive cells. A, Serum-starved human U118 glioma cells plated on coverslips were either left untreated (0 min) or treated (100 ng/ml) with FLAG-tagged soluble TWEAK (Alexis Inc.). Unbound FLAG-TWEAK was washed away and 30 min later the cells were fixed in 3% buffered formalin. Indirect immunofluorescence analysis was performed using an anti-FLAG antibody (Sigma) or control mouse IgG (eBioscience) followed by incubation with a goat anti-mouse IgG F(ab’)2 Alexa Fluor 488 secondary antibody conjugate (Molecular Probes). Photographs were taken using a fluorescent microscope. Magnification is 100X. B, Myc-tagged soluble TWEAK was purified from a stably-transfected HEK-293T cell line and added (100 ng/ml) to human endothelial cell cultures at 4 °C for 30 min. Unbound myc-TWEAK was washed away and the cells were warmed to 37 °C to allow ligand internalization. The cells were harvested at the indicated times by trypsinization and equivalent amounts of protein were subjected to SDS-PAGE and Western blot analysis using anti-myc and anti-actin antibodies. NT 30 min 30 min anti-FLAG anti-FLAG control IgG A + FLAG-TWEAK Myc B Time Since Washing Away Unbound Myc-TWEAK

TWEAK 6.25 nM 12.5 nM 25 nM 50 nM K d =3 nM GrB-TWEAK 6.25 nM 12.5 nM 25 nM 50 nM K d =8 nM A Supplementary Figure 2 B Supplementary Figure 2. Surface plasmon resonance analysis of TWEAK, GrB-TWEAK and GrB-Fc-IT4 binding to recombinant Fn14 extracellular domain. Sensograms showing binding of TWEAK (A), GrB-TWEAK (A) and GrB-Fc-IT4 (B) to Fn14 immobilized on a CM5 chip. GrB did not bind to immobilized Fn nM K d =18 nM GrB-Fc-IT4 25 nM 12.5 nM 6.25 nM

Supplementary Figure 3 MDA-MB-231 Non-treated GrB-TWEAK, 100 nM, no 1°Ab GrB-TWEAK 50 nM GrB-TWEAK 100 nM Supplementary Figure 3. GrB-TWEAK specifically internalizes into Fn14-expressing MDA-MB-231 cells. Cells were untreated or treated with 50 or 100 nmol/L GrB- TWEAK for 5 hours. The cells were fixed, acid washed to remove surface-bound material, permeabilized, and immunostained for the presence of GrB (green). The cells were counterstained with propidium iodide (red) to identify nuclei and visualized using a confocal (Zeiss LSM 510) microscope.

Supplementary Figure 4. Effect of GrB-TWEAK in combination with 5-FU, Cisplatin, GemZAR and Doxorubicin on MDA-MB-231 cells. Cells (2,000 cells/well) were seeded in 96- well plates and treated with ~ IC 25 doses of GrB-TWEAK (10 nM for MDA-MB-231) and with various concentrations of each chemotherapeutic agent as described in Supplementary Materials and Methods. After 72 h, cell viability was assessed. Normalized isobolograms were then generated using the CalcuSyn software, depicting CI values of combination drug studies. CI 1 indicate synergism, additive interaction, and antagonism, respectively. No difference in sensitivity to GrB-TWEAK was observed based on the order of treatment. Data shown is from pre-treatment of cells with chemotherapeutic agents prior to addition of GrB-TWEAK. All experiments were performed in triplicate. Supplementary Figure 4

Supplementary Figure 5 Supplementary Figure 5. Caspase-independent PARP cleavage. Pre-incubation of MDA-MB-231 and HT-29 cells with z-VAD-fmk (50 μM) for 1 h prior to 100 nM GrB- TWEAK treatment for 2 hours did not fully block PARP cleavage. 50 µM z-VAD-fmk 100 nM GrB-TWEAK PARP β-actin MDA-MB-231HT-29 Cleaved PARP

PI-9 Fn14 β-actin MDA-MB-435 AAB-527 T-24 MDA-MB-231 HT-29 HT-1080 SKOV3 ME-180 N-87 PC-3 U87-MG WM35 Calu-3 eB1 PI-9 (longer exposure) Fn14 β-actin U87 N87 HT-29 MDA-MB-231 AsPc1 Capan-1 Capan-2 L3.6P1 HCC827 A549 H358 HCC1703 H2073 H1975 PI-9 (shorter exposure) Supplementary Figure 6 Supplementary Figure 6. Expression of the GrB inhibitor proteinase inhibitor 9 (PI-9) in various cancer cell lines. Equivalent amounts of whole cell lysate were subjected to SDS- PAGE and Western blot analysis using anti-Fn14, PI-9 and β-actin antibodies.

Supplementary Figure 7 A B Supplementary Figure 7. Percent change in body weight of each group of mice from the xenograft experiments, plotted as a function of time. A. GrB or GrB-TWEAK was administered (i.v.) to mice bearing HT-29 xenograft tumors. B. Administration of GrB- TWEAK or GrB-Fc-IT4 into mice bearing MDA-MB-231/Luc breast tumor orthotopic xenografts. Arrows indicate treatment days

Supplementary Figure 8 Supplementary Figure 8. Schematic of GrB-Fc-IT4 fusion protein. GrB-Fc-IT4 is a homodimer in which an Fc domain of human IgG1 is covalently linked to GrB (N- terminus) and the anti-Fn14 humanized scFv of ITEM-4 (C-terminus). Also shown are the disulfide bridge of hinge and the approximate position of the N-linked oligosaccharides attached at Asn297 in the IgG1 Fc-domain.