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

Abstract

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.

Rare catastrophes and evolutionary legacies: human germline gene variants in MLKL and the necroptosis signalling pathway

“…They were however shown to occur in trans at 10–12 times the expected frequency in a cohort of patients suffering from chronic recurrent multifocal osteomyelitis (CRMO) [ 27 ]. While this association has not been independently replicated in a second CRMO patient cohort to date, this finding does fit nicely with mouse and human studies that implicate ‘unchecked’ necroptosis in the progression of inflammatory diseases [ 20 , 26 ].…”

“…When considering the pathophysiological impacts of necroptosis, we must not only consider genetic variation in MLKL itself , but also variation in genes that can inappropriately unleash, amplify, or even dampen the cellular activity of MLKL. Based on observations of important MLKL regulatory genes gleaned from genetically modified mice [ 20 ], we have plotted for the purposes of this review the number of disease associated human mutations reported in ClinVar [ 52 ], as accessed in September 2021 ( Figure 2A ). This tally is strongly dominated by RIPK1 , TNFAIP3 (encoding A20) and CASP8 , where several unique LOF or missense mutations are characterised by autoinflammatory and lymphoproliferative syndromes [ 53 ].…”

“…Rare mutations in human MLKL have revealed neurological and metabolic pathologies where the absence of MLKL function plays a driving role [ 23–25 ], whilst rare and de novo patient mutations in MLKL signal modulators like CASP8 and RIPK1 [ 26 ], and common polymorphisms in MLKL [ 27 ] have provided insight into pathologies where excessive necroptotic signalling is an important contributor to inflammatory disease ( Figure 2A ). Together these studies of MLKL function in humans reveal some similarities, but also many key differences to observations made in inbred laboratory mouse strains housed under specific pathogen-free conditions [ 20 ].…”

“…Mlkl gene knock-out and knock-in mutant mice have revealed necroptosis as a driver or protective factor in murine disease spanning every bodily system [ 20 ]. Simultaneously, massively parallel sequencing technologies have increased the efficiency and reduced the cost of human genotyping.…”

The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

“…In mammals, necroptosis appears to have evolved in conjunction with microbial pathogens, as there is evidence for a variety of bacterial and viral proteins that target the necroptosis pathway [ 1 – 5 ]. There is also increasing evidence implicating necroptosis in a range of inflammatory, autoimmune and neurodegenerative diseases (reviewed in [ 6 , 7 ]) and cancers (reviewed in [ 8 ]). For these reasons, there has been much interest in the cell death community in developing inhibitors of necroptosis.…”