Necroptosis has emerged as a novel physiological process of programmed cell killing that is distinct from apoptosis. Some critical components of this process have already been identified. However, in contrast to apoptosis, necroptosis is currently far less well understood and further studies to identify the critical signal transducers and control mechanisms of necroptosis are urgently needed before this process can be exploited/manipulated for therapeutic benefit in diseases such as inflammation, autoimmunity or cancer.
We hypothesise that further regulators of necroptosis, in addition to RIPK1, RIPK3 and MLKL, remain to be discovered. To address this, we used genome-wide short hairpin RNA (shRNA) knockdown (DECIPHER Pooled Lentiviral shRNA mouse library)and CRISPR/Cas9 knockout (Addgene Lentiviral mouse library) screening technologies to discover new regulators of necroptosis. We expressed these libraries in the L929 murine fibrosarcoma derived cell line that undergoes necroptosis following treatment with TNF, Smac mimetics and the broad spectrum caspase inhibitor QVD-OPH (TSQ). Gratifyingly, all known members of the necroptotic pathway (i.e. TNFR, RIPK1, RIPK3, MLKL) emerged as the highest-ranking hits. In addition, we individually cloned the MLKL shRNA from the shRNA library into a constitutive lentiviral vector that also contains a GFP reporter. We transduced L929 cells with this vector and treated them with TSQ to induce necroptosis. As expected, Mlkl shRNA-transduced cells showed marked resistance to TSQ-induced necroptosis. For the validation step candidate regulators of necroptosis were prioritised according to: a) the numbers of times that a particular shRNA or sgRNA was isolated, b) the numbers of independent shRNAs or sgRNAs targeting the same gene that were isolated and c) what is already known about the targeted gene. Overall, our findings demonstrate the reliability of our screening strategy to identify new critical components of the necroptotic machinery.