Identification of DISE-inducing shRNAs by monitoring cellular responses

Abstract

Off-target effects (OTE) are an undesired side effect of RNA interference (RNAi) caused by partial complementarity between the targeting siRNA and mRNAs other than the gene to be silenced. The death receptor CD95 and its ligand CD95L contain multiple sequences that when expressed as either si-or shRNAs kill cancer cells through a defined OTE that targets critical survival genes. Death induced by survival gene elimination (DISE) is characterized by specific morphological changes such as elongated cell shapes, senescence-like enlarged cells, appearance of large intracellular vesicles, release of mitochondrial ROS followed by activation of caspase-2, and induction of a necrotic form of mitotic catastrophe. Using genome-wide shRNA lethality screens with eight different cancer cell lines, we recently identified 651 genes as critical for the survival of cancer cells. To determine whether the toxic shRNAs targeting these 651 genes contained shRNAs that kill cancer cell through DISE rather than by silencing their respective target genes, we tested all shRNAs in the TRC library derived from a subset of these genes targeting tumor suppressors (TS). We now report that only by monitoring the responses of cancer cells following expression of shRNAs derived from these putative TS it was possible to identify DISE-inducing shRNAs in five of the genes. These data indicate that DISE in general is not an undefined toxic response of cells caused by a random OTE but rather a specific cellular response with shared features that points at a specific biological function involving multiple genes in the genome.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/186890 doi: bioRxiv preprint first posted online Sep. 10, 2017; ! 3! Introduction RNA interference is a widely used tool to reduce the expression of mRNAs. RNAi is initiated by double-stranded (ds)RNAs or pre-microRNAs, which are cleaved by Dicer, an RNase III enzyme, producing 21-23 nucleotide short interfering (si)RNAs or micro (mi)RNAs respectively, containing 2nt 3' overhangs 1, 2 . The antisense (guide) strand is then loaded onto the endonuclease argonaute 2 (Ago2) in the RNA-induced silencing complex (RISC) and directs the downstream targeting events mostly through complete complementarity between positions 2-8 (the seed region) at the 5′ end of the guide strand and a matching sequence (seed match) in the 3'UTR of targeted mRNAs. [3][4][5][6] . While in case of miRNAs, the guide strand recruits the RISC to the 3' untranslated regions (UTRs) of partially complementary mRNAs to promote translation repression or mRNA cleavage 7, 8 , the guide strand of an siRNA is designed to be fully complementary to the target mRNA and directs the enzymatic cleavage of the mRNA by the Ago2 protein [9][10][11] . RNAi can be induced by either transfecting cells with siRNAs, or by introducing short hairpin (sh)RNAs in the form of expression vectors or viruses. A...

Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism

“…679 3) Are there other genes in the human genome containing toxic seed sequences? We recently 680 identified other genes in the genome that contain DISE inducing shRNAs (Patel & Peter, 2017). 681…”

Small interfering RNA s based on huntingtin trinucleotide repeats are highly toxic to cancer cells

“…We recently reported that si‐ and shRNAs derived from CD95, CD95L , and other genes in the human genome kill cancer cells through RNAi by targeting a network of critical survival genes . DISE (death induced by survival gene elimination) was found to involve simultaneous activation of multiple cell death pathways, and cancer cells have a hard time developing resistance to this form of cell death .…”