ORIGINAL ARTICLE Genome-wide lentiviral shRNA screen identifies serine/ arginine-rich splicing factor 2 as a determinant of oncolytic virus activity in breast cancer cells ST Workenhe 1 , T Ketela 2 , J Moffat 2 , BP Cuddington 1 and KL Mossman 1 Oncolytic human herpes simplex virus type 1 (HSV-1) shows promising treatment efficacy in late-stage clinical trials. The anticancer activity of oncolytic viruses relies on deregulated pathways in cancer cells, which make them permissive to oncolysis. To identify pathways that restrict HSV-1 KM100-mediated oncolysis, this study used a pooled genome-wide short hairpin RNA library and found that depletion of the splicing factor arginine-rich splicing factor 2 (SRSF2) leads to enhanced cytotoxicity of breast cancer cells by KM100. Serine/arginine-rich (SR) proteins are a family of RNA-binding phosphoproteins that control both constitutive and alternative pre-mRNA splicing. Further characterization showed that KM100 infection of HS578T cells under conditions of low SRSF2 leads to pronounced apoptosis without a corresponding increase in virus replication. As DNA topoisomerase I inhibitors can limit the phosphorylation of SRSF2, we combined a topoisomerase I inhibitor chemotherapeutic with KM100 and observed synergistic anticancer effect in vitro and prolonged survival of tumor-bearing mice in vivo. Oncogene advance online publication, 10 August 2015; doi:10.1038/onc.2015.303 INTRODUCTION Oncolytic viruses (OVs) are naturally oncotropic or engineered viruses that selectively infect and kill cancer cells without affecting the overall health of the patient. 1 OVs are biological therapies that are directly cytotoxic to tumor cells, capable of replicating in hypoxic tumor microenvironments and able to provoke robust antitumor immune responses. 2 To improve the selective replication of human herpes simplex virus type 1 (HSV-1) in tumor cells, viral proteins that are essential for replication in normal cells but dispensable in tumor cells are deleted. 3 We have developed and tested the efficacy of HSV-1 deleted for the viral protein ICP0 as oncolytic vectors. 4–6 These OVs show modest in vitro oncolysis mainly in cancer cell types with impaired abilities to mount anti-viral responses. 4 Studies in preclinical murine tumor models showed that the antitumor activity of the prototype HSV-1 ICP0-null OV KM100 depends on whether the tumor model is tolerized. 4,6 Although in vitro tumor cell cytotoxicity mirrors the in vivo therapeutic efficacy of some OVs, 7 we fail to observe a similar correlation with our herpesvirus-based OVs, 5,6,8 despite the requirement for replica- tion of competent vectors. 4 Thus, the complex molecular interactions that dictate permissivity of oncolytic HSVs in cancer cells remain elusive. The overall premise of this study was to identify factors that restrict KM100-mediated oncolysis using a genome-wide lentivirus short hairpin RNA (shRNA) screen. Primary and secondary validation screens confirmed that knockdown of serine/arginine-rich (SR) splicing factor 2 (SRSF2) consistently promotes KM100-mediated cytotoxicity via induction of apoptosis. SRSF2 belongs to a family of RNA-binding proteins essential for spliceosome assembly during constitutive and alternative splicing of messenger RNA precursors. 9 Although the spliceosome is the catalytic machine for the process of splice site selection, intron removal and exon ligation, SR proteins are required for the correct recognition of exon–intron boundaries. 9 Moreover, members of the SR family, such as SRSF1, are involved in mammalian target of rapamycin (mTOR) signaling and regulation of cell death. 10 In response to environmental cues such as virus infection, mTORC1 signals to downstream target proteins S6K and 4EBP1, which regulate ribosomal biogenesis and translational control, respectively. 11 Unlike many RNA and DNA viruses, at the early stages of a productive infection, HSV-1 induces higher levels of phosphorylated 4EBP1, allowing the assembly of eIF4F and translational progression. 12,13 In this study, we show that in the absence of SRSF2, breast cancer cells display impaired translational responsiveness during KM100 infection. SRSF2 is a nuclear-resident SR protein phosphorylated by DNA topoisomerase I. Thus, chemotherapeutics that inhibit DNA topoisomerase I strongly inhibit the phosphorylation of SRSF2. 14–18 Here, we report that pretreatment of cells with topoisomerase I inhibitors limits SRSF2 phosphorylation, leading to synergistic killing of OV-infected breast cancer cells. The in vitro anticancer activity of the combination treatment was applied in vivo, where it extended the survival of tumor-bearing mice by enhancing apoptotic tumor cell death. Thus, this study shows a novel mechanism by which oncolytic virotherapy can be improved for breast cancer treatment. 1 Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, Institute for Infectious Disease Research, McMaster University, Hamilton, ON, Canada and 2 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. Correspondence: Dr ST Workenhe or Dr KL Mossman, Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, Institue for Infectious Disease Research, McMaster University, 1280 Main Street West, MDCL 5026, Hamilton, ON, Canada L8S 4K1. E-mail: samuelworkenhe@gmail.com or mossk@mcmaster.ca Received 22 December 2014; revised 23 June 2015; accepted 9 July 2015 Oncogene (2015), 1 – 10 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc