Abstract 1345: ATM kinase activity is dispensable in mitochondrial autophagy

Abstract

Abstract Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor with protein kinase activity, is frequently deleted or mutated in human cancers including mantle cell lymphoma (MCL). Loss of ATM protein is associated with accumulation of nonfunctional mitochondria and defective mitophagy, in both murine thymocytes and in non-cancer cells, however, the molecular mechanism of ATM kinase in cancer cell mitophagy is unknown. Here, we provide evidence that mitophagy in B-Cell Lymphoma including MCL (Granta-519, Jeko-1 and Mino), HeLa and other cancer cell lines is dependent on ATM but independent of its kinase function. Routine ATM kinase activity was performed by FACS analysis following either neocarzinostatin or IR-induced PE-ATMSer1981 and FITC-H2AXSer139 phosphorylations status and reconfirmed by immunoblot assay by probing with phospho-ATMSer1981and ATM targets Kap1Ser824 or Smc1Ser966 phosphorylations. Stable knockdown of ATM in ATM proficient MCL cell lines conferred resistance to mitophagy and was associated with reduced ATP production, oxygen consumption, and increased mitochondrial ROS. ATM protein interacts with the E3 ubiquitin ligase Parkin in a kinase-independent manner in multiple cancer cell lines including MCL and HeLa, while stable knockdown of ATM in HeLa cells (Kd HeLa) resulted in proteasomal degradation of GFP-Parkin which was rescued by the proteasome inhibitor, MG132 suggesting that ATM-Parkin interaction is important for Parkin stability. Confocal analysis revealed the presence of extra-nuclear ATM in both cytoplasm and mitochondrial compartments in HeLa cells and CCCP-induced mitophagy resulted in significantly higher mitochondrial ATM-Tom20 colocalization compared to DMSO control. Moreover both cell fractionation and confocal analysis revealed more GFP-Parkin translocation and loss of Tom20 expression in the mitochondrial fraction in WT cells following CCCP treatment. In contrast, mitochondrial GFP-Parkin expression was undetectable in Kd HeLa cells indicating a specific defect in Parkin translocation in the absence of ATM during CCCP-induced mitophagy. Further, confocal analysis with anti-DNA antibody indicated significantly higher mass of mitochondrial nucleoids in Kd HeLa cells compared to WT controls suggesting that loss of ATM promotes mtDNA accumulation. Neither loss of ATM kinase activity in primary B cell lymphomas nor inhibition of ATM kinase by KU60019 in MCL, non-tumor A-T fibroblasts and HeLa cell lines rescued FCCP or CCCP-induced mitophagy signifying that ATM catalytic activity is dispensable for mitophagy. Malignant primary B-cell lymphomas including MCL without detectable ATM, Parkin, Pink1, and Parkin-Ubser65 phosphorylation were resistant to mitophagy. This data provides the first molecular evidence of the role of ATM kinase function in mitophagy in cancer. Citation Format: Aloke Kumar Sarkar, Varsha Gandhi. ATM kinase activity is dispensable in mitochondrial autophagy [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 1345.