Distinct execution modes of a biochemical necroptosis model explain cell type-specific responses and variability to cell-death cues

Necroptosis is a form of regulated cell death that has been associated with degenerative disorders, autoimmune processes, inflammatory diseases, and cancer. To better understand the biochemical mechanisms of necroptosis cell death regulation, we constructed a detailed biochemical model of tumor necrosis factor (TNF)-induced necroptosis based on known molecular interactions. Intracellular protein levels, used as model inputs, were quantified using label-free mass spectrometry, and the model was calibrated using Bayesian parameter inference to experimental protein time course data from a well-established necroptosis-executing cell line. The calibrated model accurately reproduced the dynamics of phosphorylated mixed lineage kinase domain-like protein (pMLKL), an established necroptosis reporter. A dynamical systems analysis identified four distinct modes of necroptosis signal execution, which can be distinguished based on rate constant values and the roles of the deubiquitinating enzymes A20 and CYLD in the regulation of RIP1 ubiquitination. In one case, A20 and CYLD both contribute to RIP1 deubiquitination, in another RIP1 deubiquitination is driven exclusively by CYLD, and in two modes either A20 or CYLD acts as the driver with the other enzyme, counterintuitively, inhibiting necroptosis. We also performed sensitivity analyses of initial protein concentrations and rate constants and identified potential targets for modulating necroptosis sensitivity among the biochemical events involved in RIP1 ubiquitination regulation and the decision between complex II degradation and necrosome formation. We conclude by associating numerous contrasting and, in some cases, counterintuitive experimental results reported in the literature with one or more of the model-predicted modes of necroptosis execution. Overall, we demonstrate that a consensus pathway model of TNF-induced necroptosis can provide insights into unresolved controversies regarding the molecular mechanisms driving necroptosis execution for various cell types and experimental conditions. ### Competing Interest Statement The authors have declared no competing interest.