Juliane Daggett-Vondras

Combination of selinexor and oncolytic MYXV significantly reduced viability of human cancer cells. PANC-1 (A–D) and Colo205 (E–H) cells in 96-well plates were left untreated (mock) or treated with different concentration of selinexor, or infected with different MOI of MYXV, or treated with a combination of selinexor and MYXV for indicated timepoints. Cell viability was measured using the MTS assay reagents. Data represent mean ± SD and n = 4.

Selinexor significantly enhanced MYXV infection and replication in 3D culture of human cancer cell lines. PANC-1 (A), MDA-MB435 (C), and HT29 (E) cells were treated with different concentration of selinexor for 1 hour and then infected with vMyx-GFP-TdTomato in the presence of selinexor. Fluorescence images were taken 72 or 96 hpi. Quantification of the level of GFP fluorescence as relative fluorescence units (RFU) from PANC-1 (B), MDA-MB435 (D), and HT29 (F) cells are shown. Data represent mean ± SD and n = 3. Statistically significant differences in comparison with infection without selinexor are indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

CRM1 knockdown significantly enhanced MYXV replication in human cancer cells. PANC-1 cells were transiently transfected with CRM1 siRNA or control NT siRNA. After 48 hours, the cells were infected with vMyx-GFP at MOI of 0.5 or 5.0 for 1 hour and replaced with fresh media. A, Images showing expression of GFP after 48 and 72 hpi. The cells were harvested at 48 and 72 hpi to determine progeny virus formation by titration assay on permissive RK13 cells. Virus titers after infection with an MOI of 0.5 (B) or 5.0 (C). Data represent mean ± SD and n = 3. Statistically significant differences in comparison with infection with virus alone are indicated. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. D, A549 cells were transfected with CRM1 siRNA and infected with vMyx-GFP [MOI = 0.1 (low MOI) or 3.0 (high MOI)]. After 24 hpi, the cells were fixed and stained with antibodies against DHX9. Nuclei were stained with DAPI and imaged by fluorescence microscope. E, Western blot analysis of CRM1 protein level in A549 cells after transfection of siRNAs for 48 hours; actin as loading control. Assays were performed in triplicate and a representative is shown.

Prolonged replication of MYXV in the tumor bed of the Selinexor treated mice.

Selinexor significantly enhances MYXV replication in PANC-1 xenograft tumors in NSG mice, reduces tumor burden and prolong survival. A, Diagram of experimental setup. NSG mice were inoculated with PANC-1 cells at day 0 via subcutaneous injection on both flanks. Animals received four doses of selinexor by oral gavage and three intratumoral injection of vMyx-FLuc on the right flank. Mice were imaged for luciferase activity 24 and 48 hours after first virus injection. B, Luminescence images taken using IVIS imaging system 24 and 72 hours after the first injection. C, Quantification of luminescence signals as relative light units (RLU) from images taken 24 and 72 hours after first injection. D–E, Tumor volumes on the left and the right flanks were measured at different days after the start of treatments. Results are presented as mean ± SEM. F, Kaplan–Meier survival curves comparing animals with different treatment groups. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Combination of selinexor and oncolytic MYXV significantly reduced human cancer cells proliferation. A, Schematic representation of the cell proliferation assay protocol using Click-iT EdU kit. B, PANC-1 cells were left untreated (mock) or treated with selinexor, vMyx-GFP or a combination of selinexor + vMyx-GFP and cell proliferation assay was performed using Click-iT EdU kit. The images were taken using a fluorescence microscope. The assays were performed in triplicate and a representative is shown. C, Quantification of the number of cells showing labeling with EdU 594 (red fluorescence). A minimum of 100 cells were used for analysis from fluorescence images taken in B. Data represent mean ± SD and n = 3. Statistically significant differences among the different treatments are indicated. **, P < 0.01; ***, P < 0.001. PANC-1 (D and E) and Colo205 (F and G) cells in 96-well plates were left untreated (mock) or treated with selinexor, vMyx-GFP, a combination of selinexor + vMyx-GFP or vMyx-M11L-KO as control and cell proliferation assay was performed using CyQUANT NF cell proliferation assay kit. Data represent mean ± SD and n = 4. Statistically significant differences among the different treatments are indicated. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Selinexor significantly enhances MYXV replication in PANC-1 xenograft tumors in NSG mice, reduces tumor burden and prolong survival. A, Diagram of experimental setup. NSG mice were inoculated with PANC-1 cells at day 0 via subcutaneous injection on both flanks. Animals received four doses of selinexor by oral gavage and three intratumoral injection of vMyx-FLuc on the right flank. Mice were imaged for luciferase activity 24 and 48 hours after first virus injection. B, Luminescence images taken using IVIS imaging system 24 and 72 hours after the first injection. C, Quantification of luminescence signals as relative light units (RLU) from images taken 24 and 72 hours after first injection. D–E, Tumor volumes on the left and the right flanks were measured at different days after the start of treatments. Results are presented as mean ± SEM. F, Kaplan–Meier survival curves comparing animals with different treatment groups. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Selinexor enhances MYXV replication in vivo in xenograft tumors in NSG mice and reduces tumor burden. A, Diagram of experimental setup. NSG mice were inoculated with Colo205 or HT29 cells at day 0 via subcutaneous injection on both flanks. Animals received three doses of selinexor by oral gavage and three intratumoral injection of vMyx-FLuc on the left flank. Mice were imaged for luciferase activity 48 hours after first and second virus injection. B, Luminescence images taken using IVIS imaging system 48 hours after the first injection. C, Quantification of luminescence signals as relative light units (RLU) from images taken 48 hours after first injection. D, Luminescence images taken using IVIS imaging system 48 hours after the second injection. E, Quantification of luminescence signals as RLU from images taken 48 hours after second injection. F–G, Tumor volumes on the left and the right flanks were measured at different days after the start of treatments. Results are presented as mean ± SEM. ns, P > 0.05; *, P < 0.05; **, P < 0.01.

LMB treatment significantly enhanced MYXV replication in human cancer cells by reducing the formation of DHX9 antiviral granules. PANC-1 (A) and HT29 (B) cells were treated with different concentration of LMB for 1 hour, infected with different MOI of vMyx-GFP-TdTomato for 1 hour and replaced with fresh media containing the same doses of LMB. Fluorescence images were taken 48 hpi. PANC-1 (C) and HT29 (D) cells were harvested at different timepoints to determine progeny virus production by titration assays on permissive RK13 cells. Data represent mean ± SD and n = 3. Statistically significant differences in comparison with infection without LMB are indicated. E, A549 cells were treated with the indicated concentration of LMB for 1 hour and infected with vMyx-GFP (MOI = 1.0). After 24 hpi, the cells were fixed and stained with antibodies against DHX9. Nuclei were stained with DAPI and imaged by fluorescence microscope. F, Number of cells showing strong nuclear or cytoplasmic staining of DHX9 after 24 hpi. A minimum of 100 cells were used for analysis from fluorescence images taken in E. Data represent mean ± SD and n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Combination of selinexor and oncolytic MYXV significantly reduced human cancer cells proliferation. A, Schematic representation of the cell proliferation assay protocol using Click-iT EdU kit. B, PANC-1 cells were left untreated (mock) or treated with selinexor, vMyx-GFP or a combination of selinexor + vMyx-GFP and cell proliferation assay was performed using Click-iT EdU kit. The images were taken using a fluorescence microscope. The assays were performed in triplicate and a representative is shown. C, Quantification of the number of cells showing labeling with EdU 594 (red fluorescence). A minimum of 100 cells were used for analysis from fluorescence images taken in B. Data represent mean ± SD and n = 3. Statistically significant differences among the different treatments are indicated. **, P < 0.01; ***, P < 0.001. PANC-1 (D and E) and Colo205 (F and G) cells in 96-well plates were left untreated (mock) or treated with selinexor, vMyx-GFP, a combination of selinexor + vMyx-GFP or vMyx-M11L-KO as control and cell proliferation assay was performed using CyQUANT NF cell proliferation assay kit. Data represent mean ± SD and n = 4. Statistically significant differences among the different treatments are indicated. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.