Volume 21, Issue 11, Pages 2043-2053 (November 2013) Triptolide-Mediated Inhibition of Interferon Signaling Enhances Vesicular Stomatitis Virus-Based Oncolysis  Fethia Ben Yebdri, Julien Van Grevenynghe, Vera A Tang, Marie-Line Goulet, Jian Hui Wu, David F Stojdl, John Hiscott, Rongtuan Lin  Molecular Therapy  Volume 21, Issue 11, Pages 2043-2053 (November 2013) DOI: 10.1038/mt.2013.187 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Triptolide (TPL) inhibits vesicular stomatitis virus (VSV)-mediated ISG induction in a dose-proportional manner. (a) HEK293 cells were transfected with ISRE-Luc reporter and treated with TPL (2.5, 5.0, and 10.0 nmol/l) or vehicle. Cells were left untreated or were challenged with SeV (40 HAU/ml) or stimulated with interferon (IFN)-α (1,000 U/ml). Luciferase activity was analyzed 24 hours posttransfection and fold activation was determined compared with control. (b) HEK293 cells were treated with 50 nmol/l of TPL and left untreated or were infected with SeV (40 HAU/ml) or stimulated with IFN-α (1,000 U/ml). Western blotting was performed to assess expression of ISG-56 (top panel), RIG-I (middle panel), and β-actin (bottom panel) proteins using ISG56, RIG-I, and β-actin antibodies. (c) Human androgen-independent prostate cancer cell line PC3 cells were preincubated with TPL (12.5, 25.0, and 50.0 nmol/l) or vehicle and infected with VSV-Δ51 (0.005 MOI). The expression of ISGs was assessed using quantitative real-time PCR analysis of total RNA isolated from PC3 cells. (d) Whole-cell extracts (WCEs) were analyzed by immunoblotting with anti-ISG56 (left top panel), anti-PY701 STAT1 (left second panel), anti-ISG15 (left third panel), anti-IRF7 (right top panel), and anti-β-actin (right and left bottom panels) antibodies. Molecular Therapy 2013 21, 2043-2053DOI: (10.1038/mt.2013.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Triptolide (TPL) inhibits IFN signaling downstream of IRF3 activation at the transcription level. (a) HEK293 cells were transfected with ISRE-Luc reporter along with vector, TRIF, RIG-IN, MAVS, TBK1, or IRF3(5D) expression plasmid. Cells were left untreated or were treated with TPL (2.5, 5.0, and 10.0 nmol/l). Luciferase activity was monitored 24 hours posttransfection. (b) PC3 cells were either treated with 12.5, 25.0, and 50.0 nmol/l of TPL or not treated and infected with VSV-Δ51 (0.005 MOI) for 24 and 48 hours. Whole-cell extracts were analyzed by immunoblotting with anti-p-S396-IRF3 (top panel), anti-IRF3 (middle panel), and anti-β-actin (bottom panel) antibodies. (c) PC3 cells were treated with TPL, flavopiridol, or α-amanitin and challenged with VSV. ISG56 (top panel), RIG-I (middle panel), and β-actin (bottom panel) expression was analyzed by western blot. Molecular Therapy 2013 21, 2043-2053DOI: (10.1038/mt.2013.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Triptolide (TPL) enhances vesicular stomatitis virus (VSV) viral replication in PC3 and other resistant cell lines in a dose- and time-dependent manner. PC3 cell line was either pretreated or not with indicated doses of TPL for 30 minutes (TPL was present in the medium). Cells were infected with VSV-Δ51-GFP (0.005 MOI) for 1 hour. The images were captured at 48 hours postinfection by fluorescent microscopy (Axiovert 40 CFL, Zeiss, Thornwood, NY) at a (a) original magnification ×10. Then, 24 and 48 hours postinfection, percentage of infected cells was determined by (b and c) flow cytometry analysis of GFP expression. VSV mRNA expression was examined by quantitative real-time PCR and the VSV mRNA level is displayed (d) as fold expression relative to the untreated VSV-infected sample. Virus released from infected cells was measured by (e) plaque assay and (f) western blotting for measuring viral proteins using anti-VSV antibody was also performed. DU145 and Karpas-422 cell lines were pretreated as described above. Cells were infected with VSV-Δ51-GFP (0.005 MOI for DU145 and 1 MOI for Karpas-422) for 1 hour. Then, 24 and 48 hours postinfection, surface expression of (g) VSV on DU145 was assessed by flow cytometry. Virus released from Karpas-422 infected cells was measured by (h) plaque assay. Histograms shown are representative of five independent experiments, and the values represent the means ± SEM for three to five independent experiments. For all the bar graphs, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 when compared with untreated VSV-infected cells. Molecular Therapy 2013 21, 2043-2053DOI: (10.1038/mt.2013.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Triptolide (TPL) enhances vesicular stomatitis virus (VSV) oncolytic activity in VSV-resistant cell lines. PC3 were preincubated with or without TPL (3.125–50 nmol/l) for 30 minutes and then infected with VSV-Δ51-GFP. Induction of apoptosis was monitored 24 and 48 hours postinfection by (a and b) flow cytometry using annexin-V-allophycocyanin. DU145 and Karpas-422 cell lines were treated as described above. Induction of apoptosis in DU145 was monitored 24 and 48 hours postinfection by (c) flow cytometry using annexin-V-allophycocyanin. The levels of annexin-V binding and propidium iodide (PI) were also measured on (d) VSV-infected Karpas-422. Histograms shown are representative of five independent experiments, and the graphs represent the mean ± SEM of at least three independent experiments. Each value for TPL plus VSV was compared with vehicle control. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; TPL alone was compared with NI. NI denotes non–TPL-treated and non–VSV-infected samples. §P ≤ 0.05; §§P ≤ 0.01; §§§P ≤ 0.001; VSV-infected cells were compared with NI. †P ≤ 0.05; ††P ≤ 0.01. Molecular Therapy 2013 21, 2043-2053DOI: (10.1038/mt.2013.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Triptolide (TPL) enhanced vesicular stomatitis virus (VSV) apoptosis in PC3 cell line through caspase-3 activation and a decrease in Bcl-2 expression. PC3 cells were pretreated with or without TPL and then infected with VSV-Δ51-GFP as described above. The level of apoptosis was measured by flow cytometry using (a) cleaved caspase-3-PE (CC3) and (d and e) Bcl-2-V450 antibodies. The levels of annexin-V binding and cleaved caspase-3 were also measured at day 1 and day 2 postinfection in VSV-infected PC3 pretreated or untreated with TPL (6.25 nmol/l) and/or with ZVAD (100 μmol/l; b and c). Histograms shown are representative of at least three independent experiments and the values represent the means ± SEM. P values were calculated to compare the following: Each value for TPL plus VSV was compared with vehicle control. *P ≤ 0.05; **P ≤ 0.01. TPL alone was compared with NI. NI denotes non–TPL-treated and non–VSV-infected samples. §P ≤ 0.05; §§P ≤ 0.01. VSV-infected cells were compared with NI. ††P ≤ 0.01. VSV plus TPL value was compared with VSV plus TPL plus ZVAD. ‡P ≤ 0.05; ‡‡P ≤ 0.01. Molecular Therapy 2013 21, 2043-2053DOI: (10.1038/mt.2013.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Triptolide (TPL) increases vesicular stomatitis virus (VSV) virus replication in dose-dependent manner in xenograft models. (a) A PC3 subcutaneous xenograft tumor model was established in nude mice. Each group received VSV-Luc (1 × 107 pfu) intratumorally on day 0 and was either treated (with TPL (0.1 mg/Kg; day 0–6), intraperitoneally (IP)) or left untreated. (A) Viral replication at the tumor site was imaged using an in vivo imaging system. (b) Mice were injected with VSV-Luc (day 0) and treated (IP) with TPL or vehicle for 1 and 3 days, respectively. Five mice were killed at each time and tumors were collected to determine virus replication using luciferase (top panel) and plaque (bottom panel) assays. Mice were also injected with VSV-Luc (day 0) and treated (IP) with TPL at 0.1, 0.05, and 0.01 mg/kg. Tumors were collected 24 hours posttreatment and virus production was analyzed by luciferase assay (middle panel). (c) A murine TSA mammary adenocarcinoma cell (3 × 105 cells) subcutaneous graft model was established in immunocompetent mice. One week later (palpable tumors), animals were treated by intratumoral injection with VSV (2 × 107 pfu/dose) on day 0 and treated (IP) with TPL at 0.1, 0.05, and 0.01 mg/kg. Luciferase assay was performed to monitor virus replication 1 day after infection initiation. *P < 0.05; **P < 0.01 when compared with vehicle. Molecular Therapy 2013 21, 2043-2053DOI: (10.1038/mt.2013.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Vesicular stomatitis virus (VSV) plus triptolide (TPL) combination treatment reduces tumor progression in mouse xenograft model and prolongs survival. (a) Schematic representation of the treatment schedule for TSA xenograft model. Murine TSA (3 × 105 cells) subcutaneous graft model was established in BALB/c. Animals were treated by intratumoral injection with VSV (2 × 107 pfu/dose) on days 0 and 3 and treated (intraperitoneally) with TPL (0.1 mg/kg) or vehicle for a period of 7 days. (b) The cytotoxicity of TPL was assessed by monitoring mouse body weight. (c) The tumor growth was monitored using caliper measurements three times per week and the average tumor volumes (n = 8) are shown. (d) Cumulative survival rate was also evaluated. **P ≤ 0.01; ***P ≤ 0.001; when comparing VSV plus TPL combination treatment with single treatments and NI. NI denotes non–TPL-treated and non–VSV-infected samples. Molecular Therapy 2013 21, 2043-2053DOI: (10.1038/mt.2013.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions