Mixed lineage kinase domain-like protein (Mlkl) was recently found to interact with receptor interacting protein 3 (Rip3) and to be essential for tumor necrosis factor (TNF)-induced programmed necrosis (necroptosis) in cultured cell lines. We have generated Mlkl-deficient mice by transcription activator-like effector nucleases (TALENs)-mediated gene disruption and found Mlkl to be dispensable for normal mouse development as well as immune cell development. Mlkl-deficient mouse embryonic fibroblasts (MEFs) and macrophages both showed resistance to necrotic but not apoptotic stimuli. Mlkl-deficient MEFs and macrophages were indistinguishable from wild-type cells in their ability to activate NF-κB, ERK, JNK, and p38 in response to TNF and lipopolysaccharides (LPS), respectively. Consistently, Mlkl-deficient macrophages and mice exhibited normal interleukin-1β (IL-1β), IL-6, and TNF production after LPS treatment. Mlkl deficiency protects mice from cerulean-induced acute pancreatitis, a necrosis-related disease, but has no effect on polymicrobial septic shock-induced animal death. Our results provide genetic evidence for the role of Mlkl in necroptosis.

Necroptosis is a type of programmed cell death with great significance in many pathological processes. Tumour necrosis factor-α(TNF), a proinflammatory cytokine, is a prototypic trigger of necroptosis. It is known that mitochondrial reactive oxygen species (ROS) promote necroptosis, and that kinase activity of receptor interacting protein 1 (RIP1) is required for TNF-induced necroptosis. However, how ROS function and what RIP1 phosphorylates to promote necroptosis are largely unknown. Here we show that three crucial cysteines in RIP1 are required for sensing ROS, and ROS subsequently activates RIP1 autophosphorylation on serine residue 161 (S161). The major function of RIP1 kinase activity in TNF-induced necroptosis is to autophosphorylate S161. This specific phosphorylation then enables RIP1 to recruit RIP3 and form a functional necrosome, a central controller of necroptosis. Since ROS induction is known to require necrosomal RIP3, ROS therefore function in a positive feedback circuit that ensures effective induction of necroptosis.

Receptor-interacting protein kinase 3 (RIP3)-regulated production of reactive oxygen species (ROS) positively feeds back on tumour necrosis factor (TNF)-induced necroptosis, a type of programmed necrosis. Glutamine catabolism is known to contribute to RIP3-mediated ROS induction, but the major contributor is unknown. Here, we show that RIP3 activates the pyruvate dehydrogenase complex (PDC, also known as PDH), the rate-limiting enzyme linking glycolysis to aerobic respiration, by directly phosphorylating the PDC E3 subunit (PDC-E3) on T135. Upon activation, PDC enhances aerobic respiration and subsequent mitochondrial ROS production. Unexpectedly, mixed-lineage kinase domain-like (MLKL) is also required for the induction of aerobic respiration, and we further show that it is required for RIP3 translocation to meet mitochondria-localized PDC. Our data uncover a regulation mechanism of PDC activity, show that PDC activation by RIP3 is most likely the major mechanism activated by TNF to increase aerobic respiration and its by-product ROS, and suggest that RIP3-dependent induction of aerobic respiration contributes to pathologies related to oxidative stress.

Highlights d Prior ICT is associated with longer PFS of melanoma patients treated with MAPKi d Anti-PD-1/L1 before MAPKi combination prolongs durability of tumor regression d Targeting M2-TAMs augments and CD8 + T cells abolishes priming-associated benefit d Anti-PD1/L1 plus anti-CTLA-4 priming may further control melanoma brain metastasis

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAFnon-V600, and BRAFV600 mutations. Tumor cell–intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8+ T cells, and durable tumor regression elicited by this combination requires CD8+ T cells, which can be reinvigorated by anti–PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy. Significance: Type I RAFi + MEKi are indicated only in certain BRAFV600MUT cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8+ T cells are mechanisms that may be further exploited. This article is highlighted in the In This Issue feature, p. 521