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. 2018 Jan 31:8:71-81.
doi: 10.1016/j.omto.2018.01.001. eCollection 2018 Mar 30.

Targeting Poxvirus Decapping Enzymes and mRNA Decay to Generate an Effective Oncolytic Virus

Hannah M Burgess  1 , Aldo Pourchet  1 , Cristina H Hajdu  2   3 , Luis Chiriboga  2 , Alan B Frey  4   3 , Ian Mohr  1   3
Affiliations

Targeting Poxvirus Decapping Enzymes and mRNA Decay to Generate an Effective Oncolytic Virus

Hannah M Burgess et al. Mol Ther Oncolytics. .

Abstract

Through the action of two virus-encoded decapping enzymes (D9 and D10) that remove protective caps from mRNA 5'-termini, Vaccinia virus (VACV) accelerates mRNA decay and limits activation of host defenses. D9- or D10-deficient VACV are markedly attenuated in mice and fail to counter cellular double-stranded RNA-responsive innate immune effectors, including PKR. Here, we capitalize upon this phenotype and demonstrate that VACV deficient in either decapping enzyme are effective oncolytic viruses. Significantly, D9- or D10-deficient VACV displayed anti-tumor activity against syngeneic mouse tumors of different genetic backgrounds and human hepatocellular carcinoma xenografts. Furthermore, D9- and D10-deficient VACV hyperactivated the host anti-viral enzyme PKR in non-tumorigenic cells compared to wild-type virus. This establishes a new genetic platform for oncolytic VACV development that is deficient for a major pathogenesis determinant while retaining viral genes that support robust productive replication like those required for nucleotide metabolism. It further demonstrates how VACV mutants unable to execute a fundamental step in virus-induced mRNA decay can be unexpectedly translated into a powerful anti-tumor therapy.

Keywords: decapping; mRNA decay; oncolytic virus.

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Figures

Figure 1
Figure 1
Protein Synthesis and Accumulation in Murine Cancer Cells Infected with D9- or D10-Deficient VACV (A) Murine MBT2 bladder carcinoma, murine 4T1 breast carcinoma, or NHDFs were mock-infected (mock) or infected (MOI = 3) with WT VACV, D9-deficient VACV (ΔD9), or D10-deficient VACV (ΔD10). At 18 hours post-infection (hpi), cells were metabolically pulse labeled with [35S]Met-Cys for 30 min. Total protein was collected and separated by SDS-PAGE, and [35S]-labeled proteins were visualized by exposing the fixed, dried gel to X-ray film. Molecular mass standards (in kDa) are shown on the left. Representative radiolabeled proteins in mock-infected NHDFs that decrease in infected cells (consistent with host shut-off) are indicated (⋅). Representative radiolabeled proteins in mock-infected MBT2 or 4T1 cells that persist in infected cells are indicated (o). (B) Samples in (A) were analyzed by immunoblotting using anti-VACV polyclonal antisera as described.
Figure 2
Figure 2
Replication of VACV D9- and D10-Deficient Mutants in Murine Cancer Cells (A and B) MBT2 bladder carcinoma cells (A) or 4T1 breast carcinoma cells (B) seeded in 12-well dishes (approximately 5 × 105 cells/well) were infected (300 pfu/well) with either WT VACV, D9-deficient VACV (ΔD9), or D10-deficient VACV (ΔD10). After 48 hr, cultures were lysed by freeze thawing and the amount of infectious virus quantified by plaque assay in BSV40 cells. (C) As in (A) and (B), except murine MCA38, adenocarcinoma cells were infected (MOI = 1). Error bars represent SEM. N = 3 from three independent experiments.
Figure 3
Figure 3
Anti-tumor Activity of VACV D9- and D10-Deficient Mutants in Murine Cancer Cells (A) 4T1 breast carcinoma cells (1 × 104) in DMEM without additives were injected s.c. into the right flank of 8-week-old, female BALB/c mice. When tumors reached approximately 50 mm3 (8 to 9 days after 4T1 inoculation), they were directly injected on days 0, 3, and 6 (indicated by downward pointing arrows) with 5.4 × 106 PFU of D10-deficient (ΔD10) VACV (N = 10 mice) or an equivalent volume of virus-free control preparation (mock) from uninfected cells (N = 10 mice). Tumor size was monitored and animals were euthanized when control-treated tumors reached approximately 1,200 mm3. Tumors were measured on the indicated days, and the average normalized values reflecting relative tumor size on each day were plotted. Initial tumor volume immediately before treatment was normalized to a relative size of 1.0. Error bars indicate SEM. p values were obtained by multiple t test. ***p < 0.001; **p < 0.01. (B) As in (A) except murine MCA38 colon adenocarcinoma cells (1 × 105) were injected s.c. into the flank of 6-week-old, female C57/Bl6 mice. When tumors reached approximately 50 mm3 (approximately 7 days after MCA38 inoculation), they were directly injected on days 0, 3, and 6 with 1.0 × 106 PFU of D10-deficient (ΔD10) VACV (N = 10 mice), 1.0 × 106 PFU of D9-deficient (ΔD9) VACV (N = 10 mice), or an equivalent virus-free control preparation from uninfected cells (N = 10 mice). Tumor size was monitored and animals were euthanized when control-treated tumors reached approximately 1,200 mm3. Between day 9 and 12, three mice died in the mock-treated group, two mice died in the ΔD10-treated group, and one mouse died in the ΔD9-treated group. Error bars indicate SEM. p values were obtained by multiple t test. Gray * above the mock-treated line indicates comparisons of mock versus ΔD10. Black * below the mock-treated line indicates comparisons of mock versus ΔD9. **p < 0.05; ***p ≤ 0.005.
Figure 4
Figure 4
Anti-tumor Activity of VACV D9- and D10-Deficient Mutants in Human Tumor Xenografts in Immunocompromised Mice HepG2 human HCC cells were injected (1 × 107) s.c. into the flank of 8-week-old, female athymic, BALB/c nude mice. When tumors reached approximately 50 mm3 (approximately 7 days after HepG2 inoculation), they were directly injected on days 0, 3, 6, and 9 (indicated by downward black arrows) with 1.0 × 106 PFU of D10-deficient (ΔD10) VACV (N = 10 mice), 1.0 × 106 PFU of D9-deficient (ΔD10) VACV (N = 10 mice), or an equivalent volume of virus-free control preparation (mock) from uninfected cells (N = 10 mice). Tumors were measured on the indicated days, and the average normalized values reflecting relative tumor size on each day were plotted. Initial tumor volume immediately before treatment was normalized to a relative size of 1.0. Days on which the individual, moribund animals in the ΔD9 or ΔD10 treatment groups were sacrificed are indicated by symbols below (black + for ΔD9) or above (gray o for ΔD10) the respective data points. Error bars indicate SEM. p values were obtained by multiple t test. Gray * above the mock-treated line indicates comparisons of mock versus ΔD10. Black * below the mock-treated line indicates comparisons of mock versus ΔD9. **p < 0.05; ***p < 0.005.
Figure 5
Figure 5
Histopathology of HCC Tumors Treated with Decapping-Deficient VACV OV (A–F) On day 20 (A, C, D, and F) or day 17 (B and E) post-treatment, animals treated as in Figure 4 were sacrificed and explanted tumors were fixed in formalin. Paraffin-embedded sections were prepared and stained with H&E and evaluated by light microscopy. At low magnification (20X; A–C), the amount of residual viable tumor (HCC) in mock-treated mice (A) was substantially greater than that observed in ΔD9- (B) or ΔD10-treated mice (C). Viable HCC is highlighted between the blue lines; the red line shows a satellite tumor nodule present only in mock-treated mice (A). At high magnification (400X; D–F), viable HCC appears surrounded by a capsule composed of fibroblasts and few inflammatory cells in mock-treated mice (D). In contrast, ΔD9- (E) and ΔD10-treated (F) mice show a marked fibro-inflammatory response to the viable and necrotic HCC (left of the blue line: viable HCC; right of the blue line: fibro-inflammatory response). Tumor sections from individual mice are identified by the number at the top of the panels.
Figure 6
Figure 6
Characterization of Inflammatory Infiltrates in OV-Treated Tumors by Immunohistochemistry On day 20 (mock, ΔD10) or day 17 (ΔD9) post-treatment, tumor xenographs from animals treated as in Figure 4 were harvested, fixed, and embedded. Samples were processed for immunohistochemistry (IHC) as described in Materials and Methods and stained with either control or Ab reactive with macrophage (F4/80) neutrophils (Ly6C) or granzyme B (GrzB) as indicated. Samples from two independent tumors are shown to demonstrate typical staining achieved.
Figure 7
Figure 7
Preferential Enrichment of OV Antigens in Tumor Tissue Tumors and surrounding tissue from animals treated as in Figure 4 were harvested and fixed on day 17 (ΔD9-treated mice #67 and #69) or day 20 (mock-treated mouse #64; ΔD10-treated mice #51 and #52). Samples were processed for IHC as described in Materials and Methods, stained with antibody reactive with human NuMA (purple nuclear staining) or VACV (brown cytoplasmic staining), and counterstained with hematoxylin. From left to right, 10X and 40X magnifications are shown. 40X magnifications highlight representative fields showing the intersection of NuMA-positive cells (magenta arrowhead) with normal mouse tissue (blue arrowhead) and show that VACV antigens preferentially accumulate in and appear restricted to NuMA-positive tumor cells (white arrowhead).
Figure 8
Figure 8
Hyperactivation of PKR in Non-tumorigenic Human Cells Following Infection with D9- or D10-Deficient VACV Human HCC (HepG2, Hep3B, and Huh7) or untransformed, non-tumorigenic cBAL111 human liver cells were either mock-infected or infected (MOI = 5) with WT VACV, D9-deficient VACV (ΔD9), or D10-deficient VACV (ΔD10). After 18.5 hr, total protein was isolated and analyzed by immunoblotting with either total PKR or a PKR phospho-specific antibody as described.
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