Abstract 1941: The regulation of ATG9A-mediated autophagy by an ULK1-independent ATG13 complex

Cancer cells rely on autophagy for survival during periods of nutrient stress or chemotherapy. The activation of autophagy in cancer is thought to be regulated by AMPK-dependent activation of the ULK1 complex, consisting of FIP200, ATG13, ATG101, and ULK1. However, the regulation and dynamic cooperation of these proteins to govern autophagy is not completely understood. Our preliminary data suggest that ATG13 and ATG101 exist in an ULK1-independent complex that plays a key role in the regulation of autophagy. In an effort to identify the function of this ULK1-independent ATG13 complex, we discovered that an ULK1 binding-deficient mutant of ATG13 interacts with ATG9A, an apical regulator of autophagy. Using a split-venus approach coupled with confocal imaging, we found that ATG13, ATG101 and ATG9A interact together at sites of autophagosome assembly. This interaction between ATG13 and ATG9A is unaffected by deletion of ULK1 or the canonical ULK1 complex protein, FIP200. Furthermore, we found that CRISPR/Cas9-mediated deletion of ATG13 led to an aberrant accumulation of ATG9A puncta that colocalize with p62/SQSTM1. Importantly, this accumulation of ATG9A in ATG13 KO cells was rescued by expression of the ULK1 binding-deficient mutant of ATG13. Taken together, these data suggest that ATG9A interacts with ATG13 and ULK1 separately, and that ATG101 is part of an ULK1-independent ATG13 complex that controls the trafficking of ATG9A to p62 puncta. We are currently developing approaches to selectively isolate, identify, and characterize additional interacting partners of the ULK1-independent ATG13-ATG101 complex, which will give us new insight into the regulation and broader function of this complex in autophagy. Citation Format: Daniel Poole, Ashari Kananngara, Vajira Weerasekara, Colten McEwan, Alexandra Thornock, Misael Lazaro, Joshua Youngs, Joshua Andersen. The regulation of ATG9A-mediated autophagy by an ULK1-independent ATG13 complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1941.