Abstract 1383: Mechanisms of ATG9 mediated aggrephagy initiation

Abstract

Abstract Highly biosynthetic cancer cells produce proteins at a rate that may exceed the capacity of protein folding machinery, resulting in an accumulation of misfolded proteins that are poly-ubiquitinated and clustered into phase-separated ‘ubiquitin-rich’ condensates. These condensates are the initiation point for a form of autophagy called aggrephagy, which maintains cancer cell homeostasis by degrading the misfolded protein condensates via the lysosome. Aggrephagy is a cancer cell vulnerability, yet its mechanisms are poorly understood, particularly the mechanisms by which autophagy machinery recognizes the ubiquitin-rich condensates to initiate aggrephagy. Among 15 core autophagy proteins, ATG9A is the only multi-pass transmembrane protein and is essential for all known forms of autophagy. Recent evidence indicates that ATG9A is a lipid scramblase, in complex with ATG2A, that channels lipids to a growing autophagosome, which ultimately engulfs the ubiquitin-rich condensate. However, the mechanism of ATG9A recruitment to these condensates is not understood. Our data indicate that genetic or chemical manipulations of cells that cause ubiquitin-rich condensates to accumulate causing a corresponding accumulation of ATG9A. Furthermore, the induction of artificial ubiquitin-rich condensates, composed of chains of M1-linked ubiquitin, is sufficient to recruit ATG9A. Using a CRISPR knockout and reconstitution approach, we show that ATG9A is essential for assembling other autophagy machinery at ubiquitin-rich condensates. Together, our data support a model in which ATG9A recruitment is the initiating step for aggrephagy. Furthermore, our data suggest a signal or molecular pattern within the condensate promotes ATG9A recruitment to initiate aggrephagy. Our current work focuses on identifying features of ATG9A and components of the ubiquitin-rich condensate that cooperate to recruit ATG9A to these sites of aggrephagy initiation. Citation Format: Deshan Madhusanka, Colton McEwan, David Broadbent, Jens Schmidt, Joshua Andersen. Mechanisms of ATG9 mediated aggrephagy initiation [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 1383.