Research ArticleCancer

Dimethyl fumarate potentiates oncolytic virotherapy through NF-κB inhibition

See allHide authors and affiliations

Science Translational Medicine  24 Jan 2018:
Vol. 10, Issue 425, eaao1613
DOI: 10.1126/scitranslmed.aao1613
  • Fig. 1 DMF promotes viral infection.

    (A) Structure of DMF. (B) VSVΔ51-resistant human renal cancer cell line 786-0 was pretreated with DMF or left untreated for 4 hours and subsequently infected with VSVΔ51 (MOI, 0.01). Corresponding viral titers were determined 48 hours after infection from supernatants [n = 3; mean ± SD; *P < 0.05, ***P < 0.001, one-way analysis of variance (ANOVA), as compared to the untreated condition]. PFU, plaque-forming units. (C and D) Various human and murine cell lines were pretreated with DMF (150 to 250 μM) or left untreated for 4 hours and subsequently infected with VSVΔ51 (MOI, 0.01). Twenty-four hours after infection, fluorescence images were taken from the infected cancer cells, as shown in (C). Corresponding viral titers were determined 48 hours after infection from supernatants, as shown in (D) (n = 3 to 4; mean ± SD; P < 0.05, two-tailed t test, as compared to the untreated counterpart for each cell line). (E and F) 786-0 cells were pretreated with DMF (150 μM) or left untreated for 4 hours and subsequently infected with (E) Sindbis virus (MOI, 10) or (F) HSV-1 (MOI, 0.01). Corresponding viral titers in supernatants were determined 48 hours after infection (n = 3; mean ± SD; **P < 0.01, ***P < 0.001, two-tailed t test, as compared to the untreated counterpart). (G) Human A549 cells were pretreated as in (B) and infected with an adenovirus expressing firefly luciferase (Ad5) at an MOI of 1. Luciferase activity was measured over the course of 7 days. Results are represented as relative light units (RLU), and background is indicated by a black line (n = 3; mean ± SD; P < 0.05, two-way ANOVA from day 2 to day 5). (H) Multistep (MOI, 0.001 and 0.01) and single-step (MOI, 3) growth curves. 786-0 cells were pretreated with DMF and infected with VSVΔ51 at an MOI of 0.001, 0.01, or 3; supernatants were titered by plaque assay (n = 3; mean ± SD). (I) 786-0 cells were pretreated with DMF for 4 hours and infected with VSVΔ51 (MOI, 0.0001). An agarose overlay was added after 1 hour of infection. Fluorescence microscopy of a representative plaque 48 hours after infection is shown. The complete dose range is presented in fig. S1B. Corresponding images of Coomassie blue stain of the full wells and the graph of average plaque diameters illustrate the enhancement of the plaque diameters in the presence of DMF (n = 20; horizontal lines indicate means; *P < 0.05, ***P < 0.001, one-way ANOVA, as compared to the mock-treated counterpart). (J) 786-0, CT26WT, and B16F10 cell lines were pretreated and infected as in (B). Cell viability was assayed 48 hours after infection. Results were normalized to the average of the values obtained for the corresponding uninfected, untreated cells (n = 8; mean ± SD; ***P < 0.001, one-way ANOVA, as compared to VSVΔ51 condition).

  • Fig. 2 DMF enhances infection ex vivo and in human clinical samples.

    (A) CT26WT and B16F10 tumors were grown subcutaneously in BALB/c and C57BL/6 mice, respectively, and excised. BALB/c and C57BL/6 mouse spleens, muscles, lungs, and brain tissue were also collected and cored. Tumor and normal tissue cores were pretreated with 150 μM DMF for 4 hours and subsequently infected with 1 × 104 PFU of oncolytic VSVΔ51 expressing GFP. Twenty-four hours after infection, fluorescence images were acquired for the tumor or normal tissue cores. Representative images from each triplicate set are shown in the upper panel. Viral titers from supernatant were determined 48 hours after infection and are shown in the lower panel (n = 3 to 4; mean ± SD; ns, not significant; *P < 0.05, two-tailed t test, as compared to the untreated counterpart). (B) Indicated human tumor tissues were treated with DMF for 4 hours and subsequently infected with 1 × 104 PFU of VSVΔ51 expressing GFP. Viral titers were determined 48 hours after infection (n = 3 to 4; mean ± SD; *P < 0.05, **P < 0.01, two-tailed t test, as compared to the untreated counterpart). (C) Representative fluorescence and bright-field images are shown for patient 8. (D) Patient-derived cell lines were treated with DMF for 4 hours and subsequently infected with an MOI of 0.01 of oncolytic VSVΔ51 expressing GFP. Corresponding viral titers were determined 48 hours after infection (n = 3; mean ± SD; *P < 0.05, two-tailed t test, as compared to the untreated counterpart). (E) Human normal tissue was treated as in (B) (n = 3 to 4; mean ± SD; two-tailed t test).

  • Fig. 3 FMAEs promote infection by VSVΔ51.

    (A) Structures of FAEs (DEF) and maleic acid esters (DEM and DMM). (B) Metabolism of DMF. DMF is hydrolyzed into monomethyl fumarate (MMF), which in turn is metabolized into FA. FA subsequently enters the Krebs cycle. (C) 786-0 cells or (D) CT26WT ex vivo tumor cores were pretreated with various FMAEs and analogs for 4 hours and subsequently infected with oncolytic VSVΔ51 expressing GFP at (C) an MOI of 0.01 or (D) 1 × 104 PFU. Twenty-four hours after infection, we obtained fluorescence images of the infected 786-0 cells or CT26WT tumor cores. Corresponding viral titers were determined from supernatants 48 hours after infection (n = 3; mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA, as compared to the untreated counterpart).

  • Fig. 4 DMF enhances VSVΔ51 therapeutic efficacy in syngeneic and xenograft tumor models.

    (A to D) CT26WT, B16F10, and HT29 tumor-bearing mice were treated intratumorally (IT) with the vehicle [dimethyl sulfoxide (DMSO)] or DMF [50 mg/kg (B16F10) or 200 mg/kg (CT26WT and HT29)] for 4 hours and subsequently injected with 1 × 108 PFU of oncolytic VSVΔ51 expressing firefly luciferase or the vehicle [phosphate-buffered saline (PBS)] intratumorally. The treatment was administered two or three times, as indicated by arrows in (C). Twenty-four hours post-infection (hpi), viral replication was monitored. (A) Representative bioluminescence images of mice. (B) Quantification of luminescence in photons/s (n = 10 to 18; horizontal lines indicate means; **P < 0.01, two-tailed t test, as compared to VSVΔ51-infected condition; dashed lines represent average background intensity). (C) Tumor volume (n = 9 to 15; data are mean ± SEM; SD values are indicated in table S1; *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA, comparing DMF + VSVΔ51 to DMSO alone). (D) Survival was monitored over time. Log-rank (Mantel-Cox) test indicates that the combined treatment significantly prolonged survival over VSVΔ51 alone (CT26WT: n = 10 to 13, P = 0.0008; B16F10: n = 9 to 14, P = 0.0039; HT29: n = 10 to 15, P = 0.0003). (E) Tumor volume and survival were monitored after reimplantation of CT26WT in cured and naïve mice from (D) (n = 3 to 5, mean ± SD). (F) Tumor volume was monitored after implantation of 4T1 cells in CT26WT-cured and naïve mice (n = 3, mean ± SD).

  • Fig. 5 FMAEs inhibit antiviral cytokine production and response to type I IFN.

    (A to C) Lysates of 786-0 cells treated with FMAEs and infected with VSVΔ51 expressing GFP were collected at 24 hours after infection, and RNA or protein was extracted. (A) Scatter plot showing the expression of all genes in infected 786-0 in the presence (y axis) or absence (x axis) of DMF. Blue dots represent genes up-regulated by DMF during infection, and red dots represent genes down-regulated by DMF during infection. (B) Heat map showing the expression of the differentially expressed genes belonging to the “response to virus” GO term. (C) List of top GO terms down-regulated by FMAEs during viral infection. FDR, false discovery rate. (D) Lysates of 786-0 cells, treated with DMF (150 μM) and infected with VSVΔ51 expressing GFP or left untreated, were collected at 24 hours after infection and probed for indicated proteins by Western blot. (E) 786-0 cells were pretreated for 4 hours with DMF (150 μM) or mock-treated and infected with VSVΔ51 or wtVSV (MOI, 0.01). Corresponding viral titers were determined from supernatants 48 hours after infection (n = 3; mean ± SD). (F) 786-0 cells were treated with DMF (150 μM) or MMF (1500 μM) or mock-treated for 4 hours and infected with VSVΔ51ΔG at an MOI of 1. At 12 or 16 hours after infection, supernatants were collected and used to precondition 786-0 cells for 4 hours, and then the cells were infected with VSVΔ51 or wtVSV. Corresponding viral titers were determined 24 hours after infection from supernatants (n = 3; mean ± SD). Representative fluorescence images are shown. (G and H) 786-0 cells were treated as in (A). (G) At 16 hours after infection, supernatants were collected and assayed by ELISA for IFN-β (n = 3; mean ± SD). (H) At 36 hours after infection, supernatants were collected and assayed by ELISA for IFN-α (n = 3; mean ± SD). (I) 786-0 cells were treated with FMAEs for 6 hours and with IFN-β or IFN-α for 4 hours and subsequently infected with VSVΔ51 at an MOI of 0.1. Corresponding viral titers were determined from supernatants 48 hours after infection (n = 3; mean ± SD). (J) 786-0 cells were pretreated with 200 μM DMF for 1 or 4 hours and treated with IFN-β (250 U/ml) for 30 min or left untreated. Cell lysates were extracted and probed for pSTAT1, STAT1, and β-actin by Western blot.

  • Fig. 6 DMF inhibits NF-κB translocation upon infection.

    (A) Structures of DMS, MMS, and S. (B and C) 786-0 cells were pretreated with the indicated DMF analogs for 4 hours and subsequently infected with oncolytic VSVΔ51 expressing GFP at an MOI of 0.01. (B) At 24 hours after infection, fluorescence images were taken from the infected 786-0 cells. (C) Corresponding viral titers were determined from supernatants 48 hours after infection (n = 3; mean ± SD; ***P < 0.001, one-way ANOVA, as compared to the untreated counterpart). (D) GSH concentrations were determined in 786-0 cells after a 4-hour treatment with FMAEs (n = 4; mean ± SD; ***P < 0.001, one-way ANOVA, as compared to the untreated counterpart). (E) 786-0 cells were grown in the presence or absence of BSO (2 mM) for 7 days and pretreated with DMF (200 μM) for 4 hours or left untreated and then infected with oncolytic VSVΔ51 expressing GFP at an MOI of 0.01. Corresponding viral titers were determined from supernatants 48 hours after infection (n = 3; mean ± SD). (F) Heat map showing the expression of the differentially expressed oxidative stress genes. Expression of genes was normalized to values obtained for untreated, uninfected control. (G) HMOX1 expression quantified by quantitative polymerase chain reaction (qPCR) from 786-0 cells after a 6-hour treatment with FMAEs (n = 3; mean ± SD). (H) siNRF2 knockdown 786-0 cells were treated with DMF or untreated and infected as in (E). Corresponding viral titers were determined from supernatants 24 hours after infection. RNA was extracted, and the expression of NRF2 and IFITM1 genes was quantified by qPCR (n = 3; mean ± SD). (I and J) Cytoplasmic and nuclear protein fractions were extracted from 786-0 cells treated with DMF (200 μM) for 4 hours and (I) subsequently infected with oncolytic VSVΔ51 expressing GFP at an MOI of 1 for 8 hours or (J) treated with TNFα (30 ng/ml) for 30 min. Cell lysates were probed for multiple proteins, as indicated, by Western blot. (K) 786-0 cells were pretreated with NF-κB inhibitors targeting IKK [IKK16 (10 μM) and TPCA1 (40 μM)] for 4 hours and subsequently cotreated with DMF (150 μM) and oncolytic VSVΔ51 expressing GFP at an MOI of 0.01. Corresponding viral titers were determined from supernatants 24 hours after infection (n = 3; mean ± SD; ***P < 0.001, one-way ANOVA, as compared to the untreated counterpart).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/425/eaao1613/DC1

    Fig. S1. DMF promotes viral spread.

    Fig. S2. FMAEs enhance VSVΔ51 infection in 786-0 and CT26WT cancer cell lines.

    Fig. S3. FMAEs enhance VSVΔ51 spread in cancer cells.

    Fig. S4. FMAEs inhibit response to type I IFN.

    Table S1. SD values for tumor volumes in Fig. 4C.

    Table S2. List of drugs, chemicals, and cytokines used in this study.

    Table S3. List of cell lines used in this study.

  • Supplementary Material for:

    Dimethyl fumarate potentiates oncolytic virotherapy through NF-κB inhibition

    Mohammed Selman, Paula Ou, Christopher Rousso, Anabel Bergeron, Ramya Krishnan, Larissa Pikor, Andrew Chen, Brian A. Keller, Carolina Ilkow, John C. Bell, Jean-Simon Diallo*

    *Corresponding author. Email: jsdiallo{at}ohri.ca

    Published 24 January 2018, Sci. Transl. Med. 10, eaao1613 (2018)
    DOI: 10.1126/scitranslmed.aao1613

    This PDF file includes:

    • Fig. S1. DMF promotes viral spread.
    • Fig. S2. FMAEs enhance VSVΔ51 infection in 786-0 and CT26WT cancer cell lines.
    • Fig. S3. FMAEs enhance VSVΔ51 spread in cancer cells.
    • Fig. S4. FMAEs inhibit response to type I IFN.
    • Table S1. SD values for tumor volumes in Fig. 4C.
    • Table S2. List of drugs, chemicals, and cytokines used in this study.
    • Table S3. List of cell lines used in this study.

    [Download PDF]

Stay Connected to Science Translational Medicine

Navigate This Article