Abstract 2529: Necrosome translocates to lysosome to induce lysosome membrane permeabilization and subsequent necroptotic cell death

Abstract

Abstract Introduction: Necroptosis is a cellular process known to be downregulated by cancer cells, and its molecular steps have been described in the literature. Mixed-lineage kinase domain like (MLKL) protein is the most terminal effector of necroptosis. Necroptosis has previously been shown to be activated by the formation of necrosome complex consisting of receptor interacting serine/threonine kinase 1 (RIPK1)-RIPK3-MLKL. MLKL is activated by RIPK3 via phosphorylation, forming phospho-MLKL (P-MLKL), leading to MLKL polymerization and cell necroptosis via lysosome membrane permeabilization (LMP) and plasma membrane rupture. We propose that the necrosome locates onto the lysosome, leading to MLKL-activation induced LMP before cell membrane rupture. Objectives: Our objective is to show that necrosome formation occurs on lysosomal membrane, which then leads to the formation of P-MLKL, and, subsequently, necroptosis via LMP and plasma membrane rupture. Methods: HT-29 cells with hemagglutinin (HA) tagged lysosomal transmembrane protein were induced to undergo necroptosis with the treatment of TNF (T), Smac-mimetic (S) and Z-VAD-FMK (Z) (abbreviated as T/S/Z). Lysosomes were extracted first by cell rupture via homogenization and then immunoprecipitation using anti-HA magnetic beads. Results: Our results suggest that LMP precedes plasma membrane permeability and that MLKL activation takes place on lysosomal membrane. Western blotting demonstrated that lysosomal cathepsin proteases, including CTSA, CTSB, CTSC, CTSD and CTSK, were released into cytosol during necroptosis, confirming lysosomal damage. Furthermore, P-RIPK1, P-RIPK3, P-MLKL and MLKL tetramer were found to be specifically associated with purified lysosomes upon necroptosis induction. Conclusions: MLKL activation on lysosomal membrane and LMP constitute an important step of necroptosis. [J.M. and R.F. contributed equally to this work.] Citation Format: Jamila Mammadova, Rojin Fatirkhorani, Shuzhen Liu, Ken Chen, Zhigao Wang. Necrosome translocates to lysosome to induce lysosome membrane permeabilization and subsequent necroptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2529.