Oncolytic viruses exploited for cancer therapy have been developed to selectively infect, replicate, and kill cancer cells to inhibit tumor growth. However, in some cancer cells, oncolytic viruses are often limited in completing their full replication cycle, forming progeny virions, and/or spreading in the tumor bed because of the heterogeneous cell types within the tumor bed. Here, we report that the nuclear export pathway regulates oncolytic myxoma virus (MYXV) infection and cytoplasmic viral replication in a subclass of human cancer cell types where viral replication is restricted. Inhibition of the XPO-1 (exportin 1) nuclear export pathway with nuclear export inhibitors can overcome this restriction by trapping restriction factors in the nucleus and allow significantly enhanced viral replication and killing of cancer cells. Furthermore, knockdown of XPO-1 significantly enhanced MYXV replication in restrictive human cancer cells and reduced the formation of antiviral granules associated with RNA helicase DHX9. Both in vitro and in vivo, we demonstrated that the approved XPO1 inhibitor drug selinexor enhances the replication of MYXV and kills diverse human cancer cells. In a xenograft tumor model in NSG mice, combination therapy with selinexor plus MYXV significantly reduced the tumor burden and enhanced the survival of animals. In addition, we performed global-scale proteomic analysis of nuclear and cytosolic proteins in human cancer cells to identify the host and viral proteins that were upregulated or downregulated by different treatments. These results indicate, for the first time, that selinexor in combination with oncolytic MYXV can be used as a potential new therapy. Significance: We demonstrated that a combination of nuclear export inhibitor selinexor and oncolytic MYXV significantly enhanced viral replication, reduced cancer cell proliferation, reduced tumor burden, and enhanced the overall survival of animals. Thus, selinexor and oncolytic MYXV can be used as potential new anticancer therapy.

Effector-triggered immunity (ETI) is a form of pathogen sensing that involves detection of pathogen-encoded virulence factors or “effectors.” To discover ETI pathways in mammals, we developed a screening approach in which we expressed individual virulence factors in a human monocyte cell line and assessed transcriptional responses by RNA sequencing. We identified a poxvirus effector, myxoma virus M3.1, which elicited an antiviral nuclear factor κB (NF-κB) response. NF-κB was unleashed by an ETI pathway that sensed M3.1 attack of two antiviral complexes: zinc finger antiviral protein and TBK1. NF-κΒ activation occurred because the proteins inhibited by M3.1—N4BP1, ZC3H12A, and TBK1—are negative regulators of NF-κB. Our study established a systematic approach for the discovery of ETI pathways, and the results illustrated how negative regulators of immune responses may function in pathogen sensing.

The nucleocytoplasmic transport of proteins using XPO1 (exportin 1) plays a vital role in cell proliferation and survival. Many viruses also exploit this pathway to promote infection and replication. Thus, inhibiting the XPO1-mediated nuclear export pathway with selective inhibitors has a diverse effect on virus replication by regulating antiviral, proviral, and anti-inflammatory pathways. The XPO1 inhibitor Selinexor is an FDA-approved anticancer drug predicted to have antiviral or proviral functions against viruses. Here, we observed that the pretreatment of cultured cell lines from human or mouse origin with the nuclear export inhibitor Selinexor significantly enhanced the protein expression and replication of mouse hepatitis virus (MHV), a mouse coronavirus. The knockdown of cellular XPO1 protein expression also significantly enhanced the replication of MHV in human cells. However, for SARS-CoV-2, Selinexor treatment had diverse effects on virus replication in different cell lines. These results indicate that XPO1-mediated nuclear export pathway inhibition might affect coronavirus replication depending on cell types and virus origin.

Selinexor treatment significantly enhanced MYXV gene expression and replication in diverse human cancer cell lines. PANC-1 (A), Colo205 (B), and MDA-MB435 (C) cells were treated with different concentration of selinexor for 1 hour, infected with different MOI of vMyx-GFP-TdTomato for 1 hour and replaced with fresh media containing the same doses of selinexor. Fluorescence images were taken 48 hpi. PANC-1 (D), Colo205 (E), and MDA-MB435 (F) cells were harvested at different times after infection 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 selinexor were observed.

Tumor burden of individual mouse received PANC-1 cells.

Oncolytic viruses exploited for cancer therapy have been developed to selectively infect, replicate, and kill cancer cells to inhibit tumor growth. However, in some cancer cells, oncolytic viruses are often limited in completing their full replication cycle, forming progeny virions, and/or spreading in the tumor bed because of the heterogeneous cell types within the tumor bed. Here, we report that the nuclear export pathway regulates oncolytic myxoma virus (MYXV) infection and cytoplasmic viral replication in a subclass of human cancer cell types where viral replication is restricted. Inhibition of the XPO-1 (exportin 1) nuclear export pathway with nuclear export inhibitors can overcome this restriction by trapping restriction factors in the nucleus and allow significantly enhanced viral replication and killing of cancer cells. Furthermore, knockdown of XPO-1 significantly enhanced MYXV replication in restrictive human cancer cells and reduced the formation of antiviral granules associated with RNA helicase DHX9. Both in vitro and in vivo, we demonstrated that the approved XPO1 inhibitor drug selinexor enhances the replication of MYXV and kills diverse human cancer cells. In a xenograft tumor model in NSG mice, combination therapy with selinexor plus MYXV significantly reduced the tumor burden and enhanced the survival of animals. Additionally, we performed global-scale proteomic analysis of nuclear and cytosolic proteins in human cancer cells to identify the host and viral proteins that were upregulated or downregulated by different treatments. These results indicate, for the first time, that selinexor in combination with oncolytic MYXV can be used as a potential new therapy.