bioRxiv preprint doi: https://doi.org/10.1101/141952; this version posted September 10, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 1 2 3 4 5 Toxic si/shRNAs that kill cancer cells 6 by targeting survival 7 8 9 William Putzbach1,6, Quan Q. Gao1,6, Monal Patel1,6, Stijn van Dongen4, Ashley Haluck- 10 Kangas1, Aishe A. Sarshad5, Elizabeth Bartom2, Kwang-Youn A. Kim3, Denise M. 11 Scholtens3, Markus Hafner5, Jonathan C. Zhao1, Andrea E. Murmann1 12 and Marcus E. Peter1,2,* 13 14 1 Department of Medicine/Division Hematology/Oncology and 2 Department of Biochemistry and 15 Molecular Genetics, 3 Department of Preventive Medicine, Feinberg School of Medicine, 16 Northwestern University, Chicago, IL 60611, USA; 4 European Bioinformatics Institute (EMBL- 17 EBI), Hinxton, Cambridge CB10 1SD, UK; 5 Laboratory of Muscle Stem Cells and 18 Regulation, NIAMS, NIH, Bethesda, MD 20892, USA. 19 20 Corresponding author: Marcus Peter, E-mail: [email protected], phone: 312-503-1291; 21 FAX: 312-503-0189. 22 23 6 Shared first authorship 24 25 Keywords: 26 RNAi, Fas, cancer, CRISPR, cell death, DISE, OTE 27 28 29 Abstract

30 Over 80% of multiple tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) 31 induce a form of cell death characterized by simultaneous activation of multiple cell death 32 pathways preferentially killing transformed and cancer stem cells. We now show these 33 si/shRNAs kill cancer cells through canonical RNAi by targeting the 3’UTR of critical survival 34 genes in a unique form of off-target effect we call DISE (death induced by survival gene 35 elimination). Drosha and Dicer deficient cells, devoid of most miRNAs, are hypersensitive to 36 DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 37 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, 38 Venus, we have identified many toxic sequences - most of them located in the open reading 39 frame of CD95L. We propose that using specific toxic RNAi-active sequences present in the 40 genome can kill cancer cells. 41 bioRxiv preprint doi: https://doi.org/10.1101/141952; this version posted September 10, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

2 42 Introduction 43 One of the most popular methods utilized to reduce gene expression in cells is RNA 44 interference (RNAi). RNAi has been used in several studies to identify genes critical for the 45 survival of human cancer cell lines (Cowley et al., 2014; Hadji et al., 2014; Hart, Brown, 46 Sircoulomb, Rottapel, & Moffat, 2014; Morgens, Deans, Li, & Bassik, 2016; Wang et al., 2015). 47 During RNAi, gene expression is inhibited by small interfering (si)RNAs, small hairpin 48 (sh)RNAs or micro (mi)RNAs. miRNAs are generated as primary transcripts in the nucleus 49 where they undergo processing to pre-miRNAs by the Drosha-DGCR8 complex before being 50 exported to the cytosol by exportin 5 (Ha & Kim, 2014; Krol, Loedige, & Filipowicz, 2010). 51 Once in the cytosol, pre-miRNAs and shRNAs are cleaved by Dicer, a type III RNase that 52 functions in complex with TRBP, generating 21-23 nucleotide long fragments of double-stranded 53 RNA (dsRNA) that have two nucleotide 3' overhangs (Zamore, Tuschl, Sharp, & Bartel, 2000). 54 DsRNA fragments or chemically synthesized double stranded siRNAs are loaded into the RNA- 55 induced silencing complex (RISC) as single stranded RNAs (the guide RNA) (Siomi & Siomi, 56 2009). A near-perfect complementarity between the guide strand of the si/miRNA and the target 57 mRNA sequence results in cleavage of the mRNA (Pratt & MacRae, 2009). Incomplete 58 complementarity results in inhibition of translation and contributes t