Heterogeneous responses to low level death receptor activation are explained by random molecular assembly of the Caspase-8 activation platform

Ligand binding to death receptors activates apoptosis in cancer cells. Stimulation of death receptors results in the formation of intracellular multiprotein platforms that either activate the apoptotic initiator Caspase-8 to trigger cell death, or signal through kinases to initiate inflammatory and cell survival signalling. Two of these platforms, the Death-Inducing Signalling Complex (DISC) and the RIPoptosome, also initiate necroptosis by building filamentous scaffolds that lead to the activation of mixed lineage kinase domain-like pseudokinase. To explain cell decision making downstream of death receptor activation, we developed a semi-stochastic model of DISC/RIPoptosome formation. The model is a hybrid of a direct Gillespie stochastic simulation algorithm for slow assembly of the RIPoptosome and a deterministic model of downstream caspase activation. The model explains how alterations in the level of death receptor-ligand complexes, their clustering properties and intrinsic molecular fluctuations in RIPoptosome assembly drive heterogeneous dynamics of Caspase-8 activation. The model highlights how kinetic proofreading leads to heterogeneous cell responses and results in fractional cell killing at low levels of receptor stimulation. It reveals that the noise in Caspase-8 activation—exclusively caused by the stochastic molecular assembly of the DISC/RIPoptosome platform—has a key function in extrinsic apoptotic stimuli recognition.Author summary: Death receptors are targets of novel cancer therapeutics. Most of them signal through flexible multiprotein platforms to either activate apoptotic or necroptotic cell death, or propagate cell survival and pro-inflammatory signals. We focused our study on the role of dynamic assembly and composition of these platforms in the initiation of cell death at the single cell level. Since the assembly is slow through the competitive nature of protein binding within the platforms core we developed a stochastic mathematical model of the death inducing signalling platform. Our model provided an explanation for delayed cell death and fractional killing upon the death receptor stimulation. Additionally, we found that the variability in the cell death response arises through the random assembly initiates a slow noise-prone ramp activation of initiator Caspase-8 spontaneously triggering the apoptotic cascade. Our computational simulations predicted high variation in the time required for cell death induction at the single cell level and highlighted a significant role of death receptor clustering in effective Caspase-8 activation. Our knowledge and data driven model captures detailed processes governing the early events of cell death initiation and can be used to guide the development of more rational combinational treatments against cancer.