Abstract 2326: Time-resolved studies of necroptosis reveal distinct regulatory functions for NFκB

Abstract

Abstract Resistance to regulated cell death (RCD) is an important hallmark of cancer and reason why chemotherapy fails. Efficient reprogramming of cancer cells requires a molecular and dynamic understanding of regulatory circuits. Research has uncovered several resistance mechanisms for apoptosis with nuclear factor κB (NFκB) as a key inhibitory transcription factor. Targeted induction of necroptosis, which was recently discovered as an alternative form of RCD, could be a promising strategy to overcome apoptosis resistance. However, whether NFκB has a regulatory role in necroptosis is less clear. Here, we develop a molecular and time-resolved understanding of how NFκB regulates necroptosis. We present a real-time experimental system to quantify necroptosis in single cancer cells to study the cytotoxic effect of substances while avoiding the bias of proliferation in endpoint protocols. Using live-cell microscopy, computational cell tracking and automated image analysis we quantitate necroptotic death rates in L929 cells, a murine fibrosarcoma cell line, treated with tumor necrosis factor (TNF). While it is known that L929 cells predominantly respond with necroptosis despite rapid activation of NFκB, our experiments reveal an unsuspected temporal phasing of necroptosis over a time course of 24 hours. By perturbing NFκB activation dynamics using targeted CRISPR/Cas9 of IκB and/or Rel proteins, we find that NFκB has at least two distinct roles regulating early and late phases of TNF-induced necroptosis. Further, we will present our molecular analysis that identified NFκB-responsive candidate genes and mapped them into a functional temporo-spatial context within the NFκB-necroptosis signaling network. Our combined approach may inform novel personalized anticancer strategies to exploit targeted necroptosis while avoiding NFκB-induced drug resistance. Citation Format: Marie Oliver Metzig, Simon Mitchell, Brooks Taylor, Alexander Hoffmann. Time-resolved studies of necroptosis reveal distinct regulatory functions for NFκB [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2326.