P051 Mapping the TNF-induced necroptosis pathway by experimental test-tube genetics in human haploid cells

Abstract

Introduction TNF signaling can result in NF-kB and MAPK activation, apoptosis or necrosis depending on cell type and cell state. Until recently necrosis has been seen as an uncontrolled mode of cell death when compared to the programmed apoptosis. The identification of RIP3 as a crucial mediator of TNF-induced necrosis clearly demonstrates that in many cases necrosis is controlled by a dedicated signaling cascade, leading to the definition of necroptosis [3] , [4] , [6] . Increasing evidences demonstrate that RIP3-mediated necroptosis participates in inflammation and in both innate and adaptive immunity. Despite this, the signaling events downstream of RIP3 kinase activation remain largely obscure. Methods Here we describe the design of a haploid genetic screen in human KBM7 cells that aims to identify new components of TNF-induced RIP3-dependent necroptosis signaling pathways. The KBM7 chronic myeloid leukemia cell line is haploid for all chromosomes except chromosome 8. This allows gene inactivation through random insertional mutagenesis using a retroviral gene-trap vector and subsequent insertion site mapping [1] , [2] . Results Our data demonstrate that the TNF signaling pathway is functional in KBM7 cells resulting in NF-kB and MAPK activation, apoptosis or necroptosis depending on defined experimental settings. Under necroptosis inducing conditions, cell death occurs without detectable caspase activation and is suppressed by the RIP1 kinase inhibitor necrostatin-1. Based on these results, mutagenized KBM7 cells will be submitted to TNF-induced necroptosis and resistant clones mapped to identify genes required for necroptotic cell death. Mixed lineage kinase domain-like protein (MLKL) has been recently shown to participate in TNF-induced necroptosis [5] . An MLKL-deficient KBM7 cell clone is completely resistant to TNF-induced necroptosis while retaining sensitivity to TNF-induced apoptosis. This finding confirms by gene inactivation in human cells that MLKL is crucial for TNF-necroptosis and, importantly, validates our screening approach. Conclusion We expect the screen to genetically define the essential molecular elements required for necroptosis. Identified components will then be characterized using affinity-purification coupled to mass spectrometry. Our study should thus contribute significantly to the molecular understanding of necroptosis, a process whose relevance in inflammation, innate and adaptive immunity has just began to be fully appreciated.