The Role of the Key Effector of Necroptotic Cell Death, MLKL, in Mouse Models of Disease

Necroptosis is an inflammatory form of lytic programmed cell death that is thought to have evolved to defend against pathogens. Genetic deletion of the terminal effector protein—MLKL—shows no overt phenotype in the C57BL/6 mouse strain under conventional laboratory housing conditions. Small molecules that inhibit necroptosis by targeting the kinase activity of RIPK1, one of the main upstream conduits to MLKL activation, have shown promise in several murine models of non-infectious disease and in phase II human clinical trials. This has triggered in excess of one billion dollars (USD) in investment into the emerging class of necroptosis blocking drugs, and the potential utility of targeting the terminal effector is being closely scrutinised. Here we review murine models of disease, both genetic deletion and mutation, that investigate the role of MLKL. We summarize a series of examples from several broad disease categories including ischemia reperfusion injury, sterile inflammation, pathogen infection and hematological stress. Elucidating MLKL’s contribution to mouse models of disease is an important first step to identify human indications that stand to benefit most from MLKL-targeted drug therapies

Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues

The necroptotic cell death pathway involves signaling through pseudokinases. Here the authors define the structural determinants of species specificity in necroptosis signaling mediated by the essential necroptotic effector pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL)

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, Mlkl, that alters the two-helix ‘brace’ that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of Mlkl homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO)

MLKL trafficking and accumulation at the plasma membrane control the kinetics and threshold for necroptosis

Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. Here the authors show that MLKL trafficking and plasma membrane accumulation are crucial necroptosis checkpoints, and that accumulation of phosphorylated MLKL at intercellular junctions promotes necroptosis

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation

Acknowledgements: We thank all the following people for their technical assistance; Aira Nuguid and Tina Cardamone (Phenomics Australia Histopathology and Slide Scanning Service- The University of Melbourne). WEHI Cytometry Facility, WEHI Antibody Facility, WEHI Centre for Dynamic Imaging, WEHI Bioservices, Cheree Fitzgibbon (WEHI), Jacinta Hansen (WEHI) Jingjing Vivian Tan and Yafei Zhang (ANU, The Australian Genomics Health Alliance). The generation of MlklS131P mice by CRISPR/Cas9 gene editing was performed by Andrew Kueh and Marco Herold (WEHI MAGEC laboratory) supported by the Australian Phenomics Network (APN) and the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. We thank Warren Alexander, Mary Speir, and Melanie Bahlo for the provision of important resources and expertize. We thank Michael Hildebrand and Tom Witkowski from Epilepsy Research Centre, Department of Medicine, Austin Health for assistance with Sanger sequencing. We are grateful to the National Health and Medical Research Council for fellowship (J.M.H., 1142669; A.L.S., 2002965; J.M.M., 1172929; J.S., 1107149), grant (J.M.M., 1105023; K.R.M., 1092602; J.S., 1105023; J.M.H., 2011584) and infrastructure (IRIISS 9000719); Arthritis Australia support to K.R.M; K.E.L funding by Future Fellowships from the ARC (FT19010266). We acknowledge scholarship support for S.E.G (Australian Government Research Training Program Stipend Scholarship; Wendy Dowsett Scholarship), Y.M (Melbourne Research Scholarship; AINSE PGRA Scholarship), D.F (Australian Government Research Training Program Stipend Scholarship), A.V.J (Australian Government Research Training Program Stipend Scholarship), and S.C (Walter and Eliza Hall Handman PhD Scholarship). Victorian State Government Operational Infrastructure Support Scheme.Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-β induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation.

Acknowledgements: We thank all the following people for their technical assistance; Aira Nuguid and Tina Cardamone (Phenomics Australia Histopathology and Slide Scanning Service- The University of Melbourne). WEHI Cytometry Facility, WEHI Antibody Facility, WEHI Centre for Dynamic Imaging, WEHI Bioservices, Cheree Fitzgibbon (WEHI), Jacinta Hansen (WEHI) Jingjing Vivian Tan and Yafei Zhang (ANU, The Australian Genomics Health Alliance). The generation of MlklS131P mice by CRISPR/Cas9 gene editing was performed by Andrew Kueh and Marco Herold (WEHI MAGEC laboratory) supported by the Australian Phenomics Network (APN) and the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. We thank Warren Alexander, Mary Speir, and Melanie Bahlo for the provision of important resources and expertize. We thank Michael Hildebrand and Tom Witkowski from Epilepsy Research Centre, Department of Medicine, Austin Health for assistance with Sanger sequencing. We are grateful to the National Health and Medical Research Council for fellowship (J.M.H., 1142669; A.L.S., 2002965; J.M.M., 1172929; J.S., 1107149), grant (J.M.M., 1105023; K.R.M., 1092602; J.S., 1105023; J.M.H., 2011584) and infrastructure (IRIISS 9000719); Arthritis Australia support to K.R.M; K.E.L funding by Future Fellowships from the ARC (FT19010266). We acknowledge scholarship support for S.E.G (Australian Government Research Training Program Stipend Scholarship; Wendy Dowsett Scholarship), Y.M (Melbourne Research Scholarship; AINSE PGRA Scholarship), D.F (Australian Government Research Training Program Stipend Scholarship), A.V.J (Australian Government Research Training Program Stipend Scholarship), and S.C (Walter and Eliza Hall Handman PhD Scholarship). Victorian State Government Operational Infrastructure Support Scheme.Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-β induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation

Abstract Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-β induced death in non-hematopoietic cells. In vivo, Mlkl S131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction

Necroptosis is a regulated form of inflammatory cell death driven by activated MLKL. Here, the authors identify a mutation in the brace region that confers constitutive activation, leading to lethal inflammation in homozygous mutant mice and providing insight into human mutations in this region