Activation of ATM kinase by ROS generated during ionophore-induced mitophagy in human T and B cell malignancies

Abstract

Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor, also possesses non-nuclear functions owing to its presence in extra-nuclear compartments, including peroxisomes, lysosomes, and mitochondria. ATM is frequently altered in several human cancers. Recently, we and others have shown that loss of ATM is associated with defective mitochondrial autophagy (mitophagy) in ataxia-telangiectasia (A-T) fibroblasts and B-cell lymphomas. Further, we reported that ATM protein but not ATM kinase activity is required for mitophagy. However, the mechanism of ATM kinase activation during ionophore-induced mitophagy is unknown. In the work reported here, using several ionophores in A-T and multiple T-cell and B-cell lymphoma cell lines, we show that ionophore-induced mitophagy triggers oxidative stress-induced ATM^Ser1981 phosphorylation through ROS activation, which is different from neocarzinostatin-induced activation of ATM^Ser1981, Smc1^Ser966, and Kap1^Ser824. We used A-T cells overexpressed with WT or S1981A (auto-phosphorylation dead) ATM plasmids and show that ATM is activated by ROS-induced oxidative stress emanating from ionophore-induced mitochondrial damage and mitophagy. The antioxidants N-acetylcysteine and glutathione significantly inhibited ROS production and ATM^Ser1981 phosphorylation but failed to inhibit mitophagy as determined by retroviral infection with mt-mKeima construct followed by lysosomal dual-excitation ratiometric pH measurements. Our data suggest that while ATM kinase does not participate in mitophagy, it is activated via elevated ROS.

Keywords: ATM kinase; Leukemia; Lymphoma; Mitophagy; ROS.

Figure 1.. ATM kinase activity is dispensable for mitophagy in A-T cells.

(A) Genomic DNA isolated from A-T cells transfected with WT or S1981A ATM plasmids and sequenced spanning mutation in exon 41 of ATM cDNA showing retention of ATM S1981A auto-phosphorylation mutation 2 weeks after G418 selection. (B) Left panel: Representative flow cytometry profile showing CCCP-induced (20 μM for 12 h) mitophagy in isogenic A-T cells (1 × 10⁶ live cells) expressing either WT or S1981A-ATM mutant constructs. DMSO served as control. Following treatments, cells were washed and stained with TMRE (PE) and MitoTracker Deep Red (APC) and acquired by FACS Calibur flow cytometer, and the data were analyzed by FlowJo software. Right panel: Superimposed histogram of APC-Mito displaying mitophagy response after CCCP treatment in A-T cells transfected with WT and S1981A plasmids. (C) Left panel: Immunoblot analysis (30 μg total protein) of isogenic A-T cells expressing WT or S1981A mutant constructs and treated with DMSO, CCCP (20 μM for 12 h), or neocarzinostatin (NCS; 40 nM for 2 h) showing p-ATM^Ser1981, p-Kap1^Ser824, p-Smc1^Ser966, and Tom20, indicating mitophagy. pcDNA control (Ctrl)-transfected A-T cells served as control. ATM and p-ATM^Ser1981 blots were probed with ECL reagent. An identical blot was cut into pieces, which were probed with the indicated antibodies. Total and phosphorylated bands were merged and shown in color for specificity in LI-COR image analysis. GAPDH was probed for loading control. Right panel: Results of densitometry analysis of Tom20 expression from the experiment shown in the immunoblot (n=5) treated with the indicated compounds (n=5). KD represents S1981A mutant ATM transfected cells. Data are presented as mean ± SEM; *p<0.05 and **p<0.01 compared with respective controls as indicated. (D) Left panel: Representative flow cytometry profile of A-T cells transfected with pcDNA control or WT or S1981A ATM plasmids. Following 2 weeks of G418 selection, cells were retrovirally infected with mKeima construct. Forty-eight hours after infection, cells were treated with DMSO, CCCP (20 μM for 12 h), or N-acetyl cysteine (NAC; 10 μM for 12 h) alone or in combination with CCCP and acquired in a Celesta flow cytometer. Lysosomal mt-mKeima was measured using dual-excitation ratiometric pH measurements at 488 nm (pH 7) and 561 nm (pH 4) and lasers with 620/29 nm and 614/20 nm emission filters, respectively. Middle panel: Relative fold change in ratio of emission at pH 4 to emission at pH 7 in different treatment groups (n=2). Right panel: Relative geomean values of mROS levels of the treated cells as determined by staining cells with MitoSOX Red mitochondrial superoxide indicator (n=2).

Figure 2.. ATM is activated by ROS produced by ionophores in multiple cancer cell lines.

(A) Left panel: Immunoblot analysis (30 μg total protein) of ATM kinase–proficient WT A-T, Jurkat (T-acute lymphoblastic leukemia), Mino (mantle cell lymphoma), and Mec-1 (chronic lymphocytic leukemia) cells treated with DMSO, CCCP (10 μM), A+O (10 μM oligomycin +4 μM antimycin A), or spermidine (50 μM) for 16 h showing ATM and p-ATM^Ser1981 and Tom20 (Li-COR image), indicating mitophagy. ATM and p-ATM^Ser1981 blots were probed with ECL reagent. The blot was cut into pieces, which were probed with the indicated antibodies. GAPDH (LI-COR image) was probed for loading control. Right panel: Results of densitometry analysis of Tom20 expression from the experiment shown in the immunoblot (n=3) showing mean ± SEM; *p<0.05, **p<0.01, and ***p<0.0001 compared with respective DMSO controls. (B) Relative geomean values of mROS levels after MitoSOX Red staining in different cell lines treated with DMSO, CCCP, A+O, or spermidine at doses identical to those in panel A (n=3 for WT A-T cells; n=4 for Mino, Jurkat, and Mec1 cells). Data are presented as mean ± SEM; *p<0.05 and **p<0.01 compared with respective DMSO controls. (C) Relative geomean values of mitochondrial mass in different cell lines after MitoTracker Deep Red staining (n=3 for WT A-T cells; n=4 for Mino, Jurkat, and Mec1 cells). Data are presented as mean ± SEM; *p<0.05 and **p<0.01 compared with respective DMSO controls. (D) Relative geomean values of mROS levels after MitoSOX Red staining (as in Fig. 2B) in different cell lines treated with DMSO, CCCP (10 μM), GSH (100 mg/ml), or both CCCP and GSH for 16 h. Data are presented as mean ± SEM (n=3); **p<0.01 compared with respective DMSO controls. (E) Relative geomean values of mitochondrial mass after MitoTracker Deep Red staining in different cell lines as in Fig. 2C. Data are presented as mean ± SEM (n=3); *p<0.05 and **p<0.01 compared with respective DMSO controls. (F) Immunoblot analysis of different ATM kinase–proficient cells lines (30 μg total protein) treated with DMSO, CCCP, GSH, or both CCCP and GSH for 16 h. The blot was cut into pieces, and the pieces were probed with the indicated antibodies. Both ATM and p-ATM^Ser1981 were probed with ECL reagent. Tom20, GAPDH, and cleaved Caspase 3 were probed using Li-COR images from separate blots. GAPDH was probed separately for loading control. (G) Immunoprecipitation analysis from Mino cell extracts treated with CCCP (100 μM for 5 h). Total cell extracts (500 μg) were pulled down with rabbit anti-ATM antibody and probed with mouse ATM antibody. Arrows indicate cleaved ATM band in the immunoprecipitate. Input (5%) of IP product was loaded and probed with indicated antibodies. Cleavage of ATM and PARP following CCCP treatment indicated double strand break–primed cell death (Li-COR images). (H) Immunoblot analysis of Mino cells (30 μg total protein) treated with DMSO, GSH, CCCP (75 μM), neocarzinostatin (NCS; 40 nM), CCCP and GSH, NCS or NCS and GSH for 90 min. The blot was cut into pieces. The p-ATM^Ser1981 blot was probed with ECL reagent; all other blots were probed with the indicated antibodies and exposed using LI-COR images.