CD95/CD95L-derived si/shRNAs kill cancer cells by silencing survival genes

Immune cells kill tumor cells by activating the CD95/Fas death receptor triggering apoptosis. Unexpectedly however, >80% of a large number of siRNAs, DsiRNAs, and shRNAs we tested targeting CD95 or CD95 ligand (CD95L) induce a unique and robust form of cell death that is characterized by simultaneous activation of multiple death pathways1 and preferentially affects transformed and cancer stem cells2. Interestingly, neither exogenous CD95L, expression of shRNA-resistant CD95 or CD95L cDNAs, nor homozygous deletion of different si/shRNA target sites using CRISPR gene editing could rescue cancer cells from this form of cell death. Analysis of RNA-seq data suggests si/shRNAs derived from CD95 or CD95L are toxic to cancer cells through RNAi by a unique off-target effect (OTE) that preferentially targets 3’UTRs of critical survival genes. We call this form of cell death DISE (Death Induced by Survival Gene Elimination). By testing 4666 shRNAs derived from the ORFs and 3’UTRs of CD95 and CD95L and an unrelated control gene, Venus, we have identified multiple DISE-inducing sequences in the RNAs of CD95 and CD95L. We also found treatment with 4 different chemotherapies induces a similar loss of survival genes, suggesting these genotoxic drugs also kill cancer cells through DISE. We hypothesize the OTE described before for RNAi could be part of a biological mechanism that regulates cancer cell death. 1. Hadji, A., et al. (2014). Death induced by CD95 or CD95 ligand elimination. Cell Reports 10, 208-222. 2. Ceppi, P., et al. (2014). CD95 and CD95L promote and protect cancer stem cells. Nature Commun 5, 5238.