Abstract 313: Essential role of ataxia telangiectasia in mitochondrial autophagy in mantle cell lymphoma

Abstract

Abstract Ataxia telangiectasia mutated (ATM) is obligatory to initiate cellular responses to DNA double-strand breaks and DNA repair to preserve genomic integrity while loss of ATM leads to increased intracellular reactive oxygen species (ROS), accumulation of aberrant mitochondria, leading to abnormal mitochondrial homeostasis thereby exasperate cancer progression. ATM is frequently altered in several human cancers including 20-50% in mantle cell lymphoma (MCL). Despite ATM is known to induce global autophagy in certain cancer cells, the role of ATM in mitochondrial autophagy (mitophagy) in human cancer is imprecise. Here, we characterize a panel of MCL cell lines for their ability to trigger mitophagy induced by the mitochondrial uncoupler CCCP/FCCP. While Granta-519 lacks a functional ATM, both Jeko-1 and Mino cells exemplify as ATM proficient. Treatment with CCCP/FCCP (75µM for 4hr) elicits rapid up-regulation in ATM ser1981 phosphorylation in both Jeko-1 and Mino cells, while Granta-519 cells are mostly non-responsive. Cells were stained with TMRE (to detect mitochondrial membrane potential) or Mitotracker deep Red (to measure total mitochondrial mass) and analyzed by flow cytometry. Cells were also stained to detect both global and mitochondrial ROS (mROS) levels. CCCP/FCCP treatment resulted in rapid loss of (depolarized) membrane potential, total mitochondria mass, mROS and global ROS in both Jeko-1 and Mino cells while Granta-519 cells maintained intermediate to higher membrane potential with relatively higher total or mROS levels compared to controls. Cell fractionation analysis revealed that a fraction of resident ATM protein is located inside the mitochondrial fraction and phosphorylated upon CCCP/FCCP treatment in both Jeko-1 and Mino cells but not in Granta-519 leading to elimination of depolarized mitochondria as revealed by their basal COX4 protein levels. Immuno-precipitation analysis revealed that CCCP/FCCP treatment in both Jeko-1 and Mino cells resulted in ATM cleavage more specifically in the mitochondrial fraction. Interestingly, ATM status is unrelated to global autophagy as judged by their respective LC3I/II processing in these cell lines. Stable lentiviral knockdown of ATM in both Jeko-1 and Mino cells resulted in defective mitophagy with relatively higher COX4 expression and mROS levels. Neocarzinostatin induced DNA damage revealed loss of ATMser1981, Kap1ser824 and p53ser15 phosphorylation compared with control shRNA reconfirmed the loss of ATM function in these cells. Human primary MCL subjects with known ATM translocation [t(11;14) n=4] retain relatively higher mitochondrial mass and mROS, and are resistant to CCCP/FCCP induced mitophagy compared with ATM proficient primary MCL cells. These data underscore the possibility that ATM plays a decisive role in modulating mitophagy and contributes to the cancer-prone phenotype observed in MCL. Citation Format: Aloke Kumar Sarkar, Kumudha Balakrishnan, Mary Ayres, Sattva S. Neelapu, Varsha Gandhi. Essential role of ataxia telangiectasia in mitochondrial autophagy in mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 313. doi:10.1158/1538-7445.AM2014-313