Abstract Ligands and DR4/5-receptor agonist antibodies such as TRAIL or Apomab trigger apoptosis in tumor cells and are currently being tested in clinical trials. Exposure of cells to these ligands induces DISC (Death receptor Induced Signaling Complex) formation. When formed, these complexes activate caspase 8 which in turn cleaves Bid and triggers mitochondrial outer membrane permeabilization (MOMP) leading to apoptosis. Although promising, drugs targeting this pathway have stalled in Phase II/III of clinical trials because of variable efficacy. Many mechanisms of resistance have been proposed but no quantitative model has been built to evaluate and compare these different hypotheses. In this study, we aim to understand which features of DISC dynamics control cell fate when exposed to DR ligands, and how genomic variations or pharmaceutical drugs affect these features and ultimately ligand sensitivity. For this task, we will build a quantitative and mechanistic model of DISC activation valid in different genetic backgrounds. Using live cell imaging and a FRET reporter of Bid cleavage, we monitored caspase 8 activity at single cell level after exposure to TRAIL. In addition, we used a MOMP reporter to determine the cells undergoing apoptosis. To identify differences between cells that survive or die, we used doses of ligands that kill only a fraction of the cell population. Using a phenomenological model, we derived parameters that characterize DISC activation based on the dynamics of the FRET reporter. These parameters are significantly different between the cells that undergo MOMP and the ones that survive. We then determined how these parameters change when using different ligands (Apomab with or without clustering agent) or in cells with different genetic backgrounds (FLIP-L/S or BCL-2 overexpression). These results define quantitative relationships between these DISC parameters, key proteins of the apoptotic pathways and cell fate. Based on our knowledge of DISC formation and caspase 8 activation, we built a detailed mechanistic model of the apoptotic pathway using PySB, a framework for rule-based modeling. We calibrated the model parameters to fit the sensitivity of DISC activation to ligand concentration and FLIP expression level. This model allowed us to predict how DISC dynamics can be pharmacologically modulated to impact cell fate, and how pharmacological perturbations of other modules of the apoptotic pathway, such as the mitochondria and effector caspases, can sensitize cells to a given ligand stimulation. In particular, our model predicts which drug combinations are effective in different genetic backgrounds, a prediction that we tested experimentally. Citation Format: Jeremie Roux, Marc Hafner, Samuel Bandara, Joshua Sims, Peter K. Sorger. Modeling the dynamics of caspase 8 activity after TRAIL stimulation reveals DISC features controlling cell fate in vitro. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C14.