Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor with protein kinase activity,is frequently altered in human cancers including mantle cell lymphoma (MCL). Loss of ATM protein is linked to accumulation of nonfunctional mitochondria and defective mitophagy, in both murine thymocytes and in A-T cells. However, the mechanistic role of ATM kinase in cancer cell mitophagy is unknown. Here, we provide evidence that FCCP-induced mitophagy in MCL and other cancer cell lines is dependent on ATM but independent of its kinase function. While Granta-519 MCL cells possess single copy and kinase dead ATM and are resistant to FCCP-induced mitophagy, both Jeko-1 and Mino cells are ATM proficient and induce mitophagy. Stable knockdown of ATM in Jeko-1 and Mino cells conferred resistance to mitophagy and was associated with reduced ATP production, oxygen consumption, and increased mROS. ATM interacts with the E3 ubiquitin ligase Parkin in a kinase-independent manner. Knockdown of ATM in HeLa cells 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. Neither loss of ATM kinase activity in primary B cell lymphomas nor inhibition of ATM kinase in MCL, A-T and HeLa cell lines mitigated FCCP or CCCP-induced mitophagy suggesting that ATM kinase activity is dispensable for mitophagy. Malignant B-cell lymphomas without detectable ATM, Parkin, Pink1, and Parkin-Ub ser65 phosphorylation were resistant to mitophagy, providing the first molecular evidence of ATM's role in mitophagy in MCL and other B-cell lymphomas.
Mitochondria are indispensable for generating the solitary cellular energy currency, namely ATP, via oxidative phosphorylation and yet they exert damaging functions in altered pathophysiological scenarios including cancer.1-5 Reactive oxygen species (ROS) emanating from mitochondria can cause inevitable damage to these organelles’ own histone-devoid circular DNA with minimum proof-reading capacity encoding 37 genes - the prerequisite of the mitochondrial DNA (mtDNA) electron transport chain. Widespread metabolic reprogramming, excessive generation of ROS due to leakage of electrons from complexes I and III of the electron transport chain,6 and dysregulation of fundamental cellular functions describe abnormal mitochondria that are structurally and functionally different from their normal counterparts.7-9 Maintenance of mitochondrial homeostasis in a cell is dependent on strict and highly dynamic mitochondrial fusion and fission cycles10,11 guided by two opposing events: reparation of damage by fusion and removal of damage by fission. In a unique scenario, a defective mitochondrion incapable of making ATP via F1-F0-ATPase, instead produces excessive amounts of ROS and is forced to consume ATP to generate membrane potential (m), impeding normal metabolic function.12 Although low levels of mitochondrial damage can be repaired by complementation through the fusion process, an excessively damaged pool of mitochondria may endanger functional mitochondria during their coexistence, affecting the quality control of mitochondria.12,13 Cells have an inherent capacity to sense damaged mitochondria and selectively degrade these defective organelles by a process called mitochondrial autophagy or mitophagy.
During mitophagy, Pink1 accumulates on the outer membrane of depolarized mitochondria14 and recruits the cytosolic ubiquitin ligase Parkin and phosphorylates both Parkin and ubiquitin, resulting in Parkin activation. Activated Parkin in turn ubiquitylates scores of outer mitochondrial membrane proteins of depolarized mitochondria followed by recruitment of multiple autophagy cargo adaptors, such as OPTN and NDP52. Finally all these cargos bind directly to LC3 in the autophagosome leading to degradation of the entire mitochondrion within autophagolysosomes.15
Ataxia telangiectasia mutated (ATM) is obligatory to initiate cellular responses to DNA double-strand breaks and DNA repair to preserve genomic integrity, and loss of ATM results in genetic disorders characterized by neurodegeneration, immunodeficiency, and cancer.16-20 ATM also possesses non-nuclear functions associated with its cytoplasmic localization in various cell types21,22 and loss of ATM leads to increased accumulation of ROS and aberrant mitochondria leading to abnormal mitochondrial homeo stasis and may trigger cancer progression.16,23 Further, loss of ATM leads to global dysregulation of ribonucleotide reductase activity and abrogation of mitochondrial biogenesis and mtDNA content.24 Cells from patients with ataxia telangiectasia (A-T) contain a greater mitochondrial mass and are defective in mitochondrial respiration compared to wild-type (WT) fibroblasts.25 However, none of the phenotypic abnormalities commonly observed in ATM-deficient cells are explained by defects in canonical DNA damage response pathways, particularly in neurodegeneration, cancer predisposition, and premature aging.
Even though ATM is frequently mutated in cancer, a comprehensive study relating ATM dysfunction in mitophagy in cancer cells and in cancer patients is lacking. Mantle cell lymphoma (MCL) is a genetically unstable and fatal B-cell non-Hodgkin lymphoma, in which deletions or inactivating mutations in the ATM gene are frequently acquired (40-75% of cases).26,27We demonstrate the role of ATM in mitophagy using MCL cell lines and primary cells obtained directly from patients, and show that ATM controls mitophagy through interaction with Parkin in a kinase-independent manner.
Cell lines and primary lymphoma analysis
Cell lines are described in the Online Supplementary Methods. Primary B-cell lymphomas were obtained following informed consent from every patient on an Institutional Review Boardapproved protocol and were in accordance to the declaration of Helsinki.
Reagents and antibodies
The reagents and antibodies used in this study are presented in Online Supplementary Table S1.
Ionizing radiation, intracellular staining and flow cytometry analysis
Ionizing radiation (IR) or neocarzinostatin was used to induce DNA damage. Two potent uncoupling agents, CCCP or FCCP, were used to induce mitophagy. Cells were stained with appropriate dyes to determine mitophagy, total and mitochrondrial ROS (mROS) and acquired by flow cytometry (FCS) assay.
Plasmids, lentiviral infection and transfection
pcDNA3.1+ Flag-His-ATM wt (#31985) and pcDNA3.1+ Flag- His-ATM kd (#31986) were obtained from Addgene. GFPParkin, GFP-LC3 and GFP vector plasmids were gifts. Cells were transfected with Lipofectamine 2000 (Invitrogen) or Fugene6 (Promega). Lentiviral non-target short hairpin (sh)RNA and Mission shATM clones and lentivirus particles were prepared according to the manufacturer’s instructions (Sigma).
Immunoprecipitation and co-immunoprecipitation experiments
Standard immunoprecipitation and co-immunoprecipitation experiments were performed by transfecting plasmids in either HEK293T, WT-HeLa or A-T cells.
Measurement of nucleoside triphosphates
Intracellular nucleotides were determined by high performance liquid chromatography analysis after perchloric acid extraction.
Oxygen consumption analyses
Cellular oxygen consumption rate was measured by a standard Seahorse assay (Seahorse Bioscience, Billerica, MA, USA).
Cell fractionation and immunoblot analysis
Cell fractionation was performed using either a NE-PER kit (Thermo Fisher) or Cell fractionation kit (Abcam; AB109719) following the manufacturers’ guidelines. Protein lysates and immunoblots were prepared and protein bands were quantified using a LI-COR Odyssey CLx Infrared Imaging System.
Quantitative reverse-transcriptase polymerase chain reaction and mitochondrial DNA analysis
Total RNA was isolated using an RNeasy Mini Kit and the reverse transcriptase reaction was performed using a RevertAidTM H Minus First Strand cDNA Synthesis kit. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using the SYBR® Green PCR Master Mix. mtDNA copy number was analyzed in total DNA by quantitative PCR (qPCR) using mtDNA- and nuclear DNA-specific primers (PMC3769921) and the copy number was calculated as described before.28
Cells were grown in chamber slides, stained with primary antibodies and processed for standard fixation for confocal analysis using an Olympus FV1000 laser confocal microscope with a 40x 1.3-oil immersion lens. All captured images were analyzed using 3I Slidebook 5.5 (SB622.214.171.124) software.
All numerical results are presented as mean ± standard error of mean. The statistical significance of differences was analyzed using a Student t-test (paired) or analysis of variance. The statistical computations were conducted with Prism (GraphPad Software, San Diego, CA, USA).
Full details of the methods are available in the Online Supplementary Methods file.
ATM-proficient and -deficient mantle cell lymphoma cell lines display differential sensitivity to FCCPinduced mitophagy.
To determine the role of ATM in mitophagy we used three MCL cell lines (Granta-519, Jeko-1 and Mino). The ATM-deficient Granta-519 line possesses a single copy of the ATM gene harboring a point mutation within the conserved residues of the kinase domain while both Jeko-1 and Mino cells are ATM-proficient.29,30 These cell lines contain the distinguishing t(11;14)(q13;q32) translocation resulting in cyclin D1 (CCND1) overexpression.31 Cells were treated with the mitochondrial uncoupler FCCP to induce mitophagy. Induction of mitophagy was associated with loss of mitochondrial membrane potential (m) in both Jeko-1 and Mino cells, while Granta-519 retained intermediate to high m and greater mitochondrial mass (Figure 1A, B). Both basal mROS and global ROS were relatively higher in Granta-519 (Figure 1C-E) compared to other MCL cell lines. Typically mitophagy is accompanied by loss of both COXIV and the mitochondrial outer membrane protein Tom20. Immunoblot analysis revealed defective FCCP-induced mitophagy in Granta-519 as COXIV and Tom20 levels were not affected by FCCP treatment while they were reduced in FCCP-treated Jeko- 1 and Mino cells (Figure 1F) with <10% cell death (Online Supplementary Figure S1A). Phosphorylation of ATMSer1981 or ATM targets, Kap1Ser824 and Smc1Ser966 was detected in both Jeko-1 and Mino cells but not in Granta-519 (Figure 1F). FCS analysis also confirmed IR-induced ATMSer1981 and H2AXSer139 phosphorylations in both Jeko-1 and Mino cells but not in Granta-519 (Figure 1G). In contrast, global autophagy induced by FCCP, as detected by LC3 lipidation, was similar in all three cell lines indicating that ATM is not required for global autophagy. Furthermore, mitophagy was not affected by the loss of p53 in Jeko-1 cells demonstrating that p53 is not required for mitophagy.
Similar experiments on human A-T isogenic cell lines revealed ATM dependency of mitophagy, as reported earlier. 23 Further analyses confirmed that while autophagy was induced by CCCP in either cell line, mitophagy was restricted to only WT cells. Furthermore, we showed that A-T cells possess relatively lower levels of Pink1 and Parkin proteins with a significantly higher basal mROS level compared with the levels in WT cells (Online Supplementary Figure S1B-F).
Cell fractionation analysis revealed the presence of ATM and phospho-ATMSer1981 in the mitochondrial fraction of both Jeko-1 and Mino cells (Figure 1H). Although the majority of ATM protein was nuclear, some ATM was also detected in the cytoplasm of these cells (Online Supplementary Figure S2A). Mitophagy was detected in both Jeko-1 and Mino cells, but not in Granta-519, as evidenced by the loss of Tom20, and was associated with both Parkin and phospho-Parkin-UBSer65 activation (Figure 1H and Online Supplementary Figure S2B). Interestingly, despite selective induction of mitophagy in both Jeko-1 and Mino cells, neither p62 recruitment nor autophagy (LC3 lipidation) was affected in ATMdeficient Granta-519 cells. Together, these data suggest that a specific defect in mitophagy, but not in global autophagy resulted from the loss of functional ATM in Granta-519 cells.
Stable knockdown of ATM elicits high mitochondrial DNA copy number and defective mitophagy in mantle cell lymphoma cell lines
To further delineate the role of ATM in mitophagy, we stably knocked down ATM in Jeko-1 and Mino cells via lentiviral shATM transduction. Immunoblot analysis confirmed loss of ATM protein in the shATM clones compared to control shRNA and were also defective in IRinduced ATMSer1981, Kap1Ser824, Smc1Ser966 and p53Ser15 phosphorylation as well as IR-induced ATMSer1981 and H2AXSer139 phosphorylation, as shown by FCS analysis (Figure 2A, B).
These shATM clones were also found to produce relatively higher mROS (Figure 2C, D), defective in FCCPinduced mitophagy and retained higher mitochondrial VDAC1, Tom20, COXIV and mtDNA copy number as compared to control shRNA. Neither, autophagy (LC3 lipidation) nor FCCP-induced loss of m was affected by ATM ablation (Figure 2E-I and Online Supplementary Figure S3A-C). Therefore, loss of ATM provokes accumulation of mROS, preservation of mitochondrial mass, and inhibition of mitophagy in human cancer cells.
Loss of ATM is associated with less mitochondrial ATP generation
Endogenous levels of ATP and UTP were significantly higher in Jeko-1 and Mino cells (Online Supplementary Figure S4A) than in Granta-519, indicating higher respiratory capacity. Further, ATM ablation in MCL cell lines resulted in significantly less ATP production (Figure 3A). This decline in ATP-linked respiration in the shATM clones resulted in depletion in the ATP pool, basal oxygen consumption rate (OCR) or in their respective stressinduced spare respiratory capacities (SRC) (Figure 3B-D). The maximal ATP output of mitochondria can be determined by addition of the mitochondrial uncoupler, FCCP, which collapses the proton gradient and generates m and triggers maximal OCR and substrate oxidation by complex IV. Moreover, SRC allows us to determine the ability of the cells to respond to increased energy demands following FCCP treatment. Thus, the observed decreases in both basal OCR and SRC in shATM clones were consistent with significant reductions in the intracellular ATP pool in ATM-depleted cells. Consistent with these findings, we found higher OCR and SRC in WT cells than in A-T cells (Online Supplementary Figure S4B,C).
ATM interacts with and confers Parkin stability in a kinase-independent manner
Having observed that ATM ablation impedes mitophagy prompted us to determine the role of ATM kinase in Parkin-mediated mitophagy in HeLa cells. ATM in WT HeLa cells was stably knocked down (Kd-ATM HeLa) by lentiviral shATM infection (Figure 4A). The distributions of global, nuclear, cytoplasmic, and mitochondrial ATM foci were quantified by confocal analysis using a cellular masking method (Online Supplementary Figure S5A). As expected, significantly more abundant ATM foci were observed in all three compartments in WT cells than in Kd-ATM cells (Figure 4B-D). CCCP treatment resulted in greater mitochondrial ATM-Tom20 co-localization than in control cells treated with dimethylsulfoxide. While both global and cytoplasmic ATM foci remained unchanged following CCCP treatment, the loss of nuclear ATM and significant gain in mitochondrial ATM likely reflects translocation of ATM from the nucleus to the mitochondria.
Confocal analysis also showed significantly higher mass of mitochondrial nucleoids in Kd-ATM HeLa cells than in WT controls (Figure 4E,F). Since HeLa cells lack detectable Parkin expression,32 both WT and Kd-ATM HeLa cells were transfected with GFP-Parkin plasmid to generate stable HeLa GFP-Parkin isogenic cell lines proficient or deficient in ATM. Surprisingly, while transient GFP-Parkin expression was fairly equal in both WT and Kd-ATM cell lines (Figure 4G), we failed to generate stable GFP-Parkinexpressing Kd-ATM HeLa cells. However, neither stable expression of GFP-vector nor GFP-LC3 was affected, suggesting a specific defect in GFP-Parkin stability in Kd-ATM HeLa cells (Figure 4H). Cell fractionation studies following exposure to CCCP (Figure 4I) revealed the presence of ATM protein in both nuclear and mitochondrial fractions in WT but not in Kd-ATM cells. GFP-Parkin expression was detected in both cytoplasm and mitochondrial fractions, and CCCP treatment enriched the abundance of mitochondrial GFP-Parkin accumulation in WT cells, resulting in a decrease in Tom20 expression via mitophagy. In contrast, mitochondrial GFP-Parkin translocation was undetectable in Kd-ATM cells following CCCP treatment. However, the cellular distribution of the endogenous autophagy adaptor protein, p62/SQSTM1, was not affected in either of these cells, suggesting that autophagy is unrelated to the loss of ATM in HeLa cells. Confocal analysis reconfirmed that CCCP significantly induced mitochondrial GFP-Tom20 double-positive foci in WT cells compared to Kd-ATM cells (Figure 4J-L). We also showed that endogenous Parkin expression is significantly lower in the majority of the early passage MCL shATM cell lines than in control shRNA-transduced cells (Figure 4M, N). These data support the notion that defective mitophagy in MCL and HeLa shATM cells is likely due to lack of Parkin stability and mitochondrial Parkin translocation.
Parkin is known to autoubiquitinate at the UBL domain and undergo proteasomal degradation in the cytosol.33 while endogenous Pink1 is known to be rapidly degraded by UBR1, UBR2 and UBR4 through the “N-end rule pathway”. 34 Interestingly, the proteasome inhibitor, MG132 rescued loss of GFP-Parkin stability in Kd-ATM HeLa cells (Figure 5A upper panel; Online Supplementary Figure S5B). Surprisingly, endogenous Pink1 expression was lower in Kd-ATM cells than in WT ones and MG132 prevented Pink1 degradation in both cell lines (Figure 5A lower panel). The kinetics of GFP-Parkin degradation following cycloheximide treatment (Figure 5B, C) suggested a significantly enhanced degradation of GFP in Kd-ATM cells than in WT HeLa cells. However, loss of ATM did not affect the stability or degradation of the long half-life (HSP90) or short half-life (MCl-1)35 proteins. Real-time RTPCR analysis from identical cells did not reveal any significant change in GFP expression (Figure 5D), arguing for a specific defect in GFP-Parkin protein stability in Kd-ATM HeLa cells.
This observation prompted us to investigate the role of ATM kinase activity in the ATM-Parkin interaction. GFPParkin- transfected WT Hela cells were immunoprecipitated with anti-ATM antibody (Figure 5E, F; Online Supplementary Figure S6A). We showed that neither ATM kinase inhibitor (KU60019) nor neocarzinostatin treatment influenced their interaction, confirming that the ATM-Parkin interaction is kinase-independent. Similarly, the endogenous interaction of ATM-Parkin in the Mino cell line was also independent of ATM kinase inhibition and KU60019 did not inhibit the interaction when cells were treated with this in combination with FCCP (Figure 5G, H; Online Supplementary Figure S6B). The efficacy of KU60019 was demonstrated by loss of ATMSer1981 and Kap1Ser824 phosphorylation (Figure 6D). In other experiments, A-T cells were co-transfected with either WT or kinase-dead (KD)-Flag-ATM and GFP-Parkin plasmids and immunoprecipitated with anti-Flag antibody. Analysis of the immunoprecipitates showed that both WT and Kd- ATM interacts with GFP-Parkin (Online Supplementary Figure S6C,D). Further immunoprecipitation analysis with anti-Parkin antibody from purified cytosolic and mitochondrial fractions from Mino cells suggests that the ATM-Parkin interaction was restricted in both these fractions (Figure 5I, J). In contrast, trypsin digestion of the mitochondrial fraction, which removes proteins from the outer membrane, revealed the presence of Parkin but not ATM in the inner membrane along with TIM23,36 a specific inner membrane translocase. These data confirm that the ATM-Parkin interaction is restricted to the outer mitochondrial membrane.
ATM kinase is dispensable in mitophagy: evidence from mantle cell lymphoma, HeLa cells and primary B-cell lymphomas
The observed kinase-independent interaction of ATMParkin led us to test the role of ATM kinase function in mitophagy using pharmacological ATM inhibitors or activators. The ATM kinase inhibitor, KU60019, failed to inhibit mitophagy in either MCL or WT A-T cells (Figure 6A-C; Online Supplementary Figure S7A, B). As expected KU60019 inhibited FCCP-induced ATMSer1981, H2AXSer139 and Kap1Ser824 phosphorylation in both MCL cell lines, but failed to rescue FCCP-induced loss of Tom20 and mitophagy (Figure 6D-F). Neither FCCP-induced Pink1 and Parkin activation, nor Parkin-UBSer65 phosphorylation was inhibited by KU60019 (Online Supplementary Figure S7C, D). Furthermore, KU60019 could not inhibit CCCPinduced mitophagy in WT HeLa cells (Figure 6G, H).
Thirty-eight primary lymphomas, including MCL (n=21), marginal zone lymphoma (MZL, n=5), follicular lymphoma (FL, n=6) and diffuse large B-cell lymphoma (DLBCL, n=6), were analyzed (Online Supplementary Table S2) for their response to FCCP-induced mitophagy. B cells isolated from healthy donors (n=3) or peripheral blood mononuclear cells (n=2) served as controls. All samples were treated with IR and their ATM kinase status was determined by FCS analysis of PE-phospho-ATMSer1981 and FITC-H2AXSer139 co-staining, while their mitophagy status was determined by FCS and immunoblot analyses (Figure 6I-K, P; Online Supplementary Figures S8A-E, S9A, D and S10A-H). Among all B-cell lymphomas screened, 13 (34%) were negative for IR-induced phospho-ATMSer1981 and H2AXSer139 activation (IR¯) (MCL=8; FL=2; DLBCL=3). Among MCL subtypes, 13 lymphomas were positive for IR-induced phospho-ATMSer1981 and H2AXSer139 activation (IR+) and eight were IR¯; no statistical correlation was observed between IR+ or IR¯ subtypes and their mitophagy status (Table 1; Figure 6K). Similarly, among all non-MCL subjects, no statistical correlation was observed between mitophagy, basal mROS and their ATM kinase status (Figure 6M; Online Supplementary Figure S9A). A higher abundance of mtDNA copy number was seen in all IR¯ MCL and non-MCL lymphomas (Figure 6L; Online Supplementary Figure S9B). Consistent with cell line data, FCCP-induced loss of m was prevalent in all primary MCL screened regardless of their IR status (Figure 6N; Online Supplementary Figure S9D). Cytogenetic analysis revealed that 18 subjects with MCL harbored the t(11;14) translocation and this was also unrelated to mitophagy (Figure 6O; Table 1; Online Supplementary Table S2). Immunoblot analysis established a strong correlation between IR-induced ATMSer1981, Kap1Ser824 and Smc1Ser966 phosphorylation, consistent with FCS analysis among all primary MCL and non-MCL lymphomas (Figure 6P; Online Supplementary Figure S10A-H).
Total and phospho ATMSer1981 protein levels were either low or undetectable in the majority of subjects with MCL compared to the levels in MCL cell lines (Jeko-1 and Mino). Both Parkin and phospho-UBSer65 Parkin levels were either low or undetectable in a subset of MCL subjects (MCL 2, 8, 15, 17, 19) (Figure 6P; Online Supplementary Figure S10A-D); IR-induced ATMSer1981, Kap1Ser824 and Smc1Ser966 phosphorylation was not detected in these lymphomas which were resistant to mitophagy. Similarly despite a lack of IR-induced ATM kinase activation, a subset of primary MCL activated phospho-Parkin-UBSer65- induced mitophagy (MCL 2, 4, 5) (Figure 6P). Conversely, analysis of basal Parkin protein expression in all primary lymphomas revealed a positive trend of higher Parkin expression among IR+ lymphomas than among IR¯ lymphomas (Figure 6Q). These data further suggest that ATM kinase activity is not required per se for phospho-UBSer65 activation-induced mitophagy in cells derived from patients.
Pink1 expression was either low or undetectable in a subset of MCL (MCL 2, 4, 8, 17, 19) (Figure 6P; Online Supplementary Figures S8B and S10A-D) and a few of these lymphomas (MCL 8, 17, 19) could not activate phospho- UBSer65 Parkin and were resistant to mitophagy. Interestingly, mitophagy was not activated in a subset of IR+ MCL (MCL 8, 13, 14, 19, 21) (Online Supplementary Figures S8B and S10A-D) suggesting a heterogeneous response. Inducible mitophagy was also prevalent among non-MCL lymphomas (Online Supplementary Figures S9A and S10E-H). Despite detectable ATM expression, IR could not activate ATMSer1981, Kap1Ser824 and Smc1Ser966 phosphorylation in a subset of DLBCL and FL (IR¯) while all MZL lymphomas were IR+ (Online Supplementary Figure S8C-E). Consistent with MCL, a few IR+ lymphomas (DLBCL4, DLBCL5, LBCL, SMZL1, FL1 and FL4) were resistant to mitophagy (Online Supplementary Figure S9A; Supplementary Table S3) while a few of the IR¯ lymphomas (DLBCL 1, 2 and FL 6) were mitophagy-proficient. No statistical correlation was observed between IR status, mROS or loss of m in these lymphomas, while mtDNA copy number (Online Supplementary Figure S9B-D) was higher in IR¯ FL and DLBCL. Although the majority of these lymphomas expressed Pink1 and Parkin, FCCP-induced phospho- UBSer65 Parkin activation was not detected in a few lymphomas (DLBCL 4, 5 and FL 2) rendering them resistant to mitophagy.
Mitophagy is a selective process of macro-autophagy eliminating intracellular pathogens and dysfunctional mitochondria by engulfing cargo into autophagolysomes37 thereby maintaining mitochondrial homeostasis, genomic stability, and integrity of cells with other healthy organelles. Therefore, imposing an opportunistic and timely exclusion of dysfunctional mitochondria via mitophagy would be useful to protect cellular and genome integrity.
ATMSer1981autophosphorylation is considered a hallmark of ATM activation38 and is important in maintaining genomic integrity.39 Recent observations suggest that ATM plays an important role in mitophagy in both murine thymocytes as well as in A-T cells23 but the mechanism is poorly understood. Parkin, a protein linked to Parkinson disease, is activated during mitophagy.40,41 These key observations raise important questions regarding a possible link between Parkin and ATM. Parkin is a tumor-suppressor protein and is known to regulate cell cycle proteins including Cyclin D1, Cyclin E, and CDK4 in cancers,42 while ATM is frequently lost and mutated in cancer thereby underscoring the need to evaluate their roles in mitophagy.
To explore the mechanism of ATM-dependent mitophagy in cancer, we selected MCL as a model system since ATM is the second most common alteration in MCL (>50%), in which ATM is frequently lost either by 11q deletion or mutation in the kinase domain and is associated with a high number of chromosomal alterations.43MCL is an aggressive form of non-Hodgkin lymphoma, and remains largely incurable; most affected patients eventually die of relapsed/refractory disease,44 thereby arguing for a need for an alternative method of treatment.
We present evidence that cancer cell mitophagy is dependent on ATM but not its kinase activity and connects Parkin in this pathway. We show that ATM is required for mitophagy but not global autophagy. Furthermore we demonstrate that ATM, but not its kinase activity, is required for ionophore-induced dissipation of m, a prerequisite signal required for Pink1-Parkin-mediated mitophagy.45 Our data are in agreement with the notion that Pink1 activation leading to Parkin activation via UBSer65 phosphorylation during mitophagy is in part ATM-dependent. We provide evidence that KU60019 failed to inhibit FCCP-induced Parkin-UBSer65 phosphorylation and mitophagy in multiple cell lines. Based on these observations, we propose that ATM kinase activity is dispensable for Parkin activation. Moreover, we observed that mitophagy inhibition following loss of ATM promotes high mROS and mtDNA copy number, low mitochondrial respiration, and ATP generation. However, none of these phenotypes clearly explains the role of ATM kinase in mitophagy.
The loss of stable GFP-Parkin levels in ATM-deficient HeLa cells suggests a novel link between ATM and mitophagy via kinase-independent, ATM-Parkin physical interactions. In normal basal condition cytosolic Parkin exists in a coiled and auto-inhibited ‘closed’ conformation and in an inactive state.46-48 Conversely Parkin ubiquitinates itself and promotes its own degradation and we demonstrated that MG132 rescued GFP-Parkin degradation in shATM HeLa cells. Consistent with this observation, we also showed ablation of ATM in MCL cell lines triggers loss of endogenous Parkin expression. These observations support the notion that ATM may play a role in conferring Parkin stability and thereby contribute to mitophagy. However, this phenomenon is in contrast to the situation in A-T cells in which Parkin is expressed in the absence of ATM, suggesting a context-specific interaction. The stability of Parkin in tumor and non-tumor cells may enforce different mechanisms. This apparent discrepancy could be due to the possibility that non-tumor A-T fibroblasts utilize an ATM-independent mechanism to stabilize Parkin.
Regardless, we show that ATM kinase activity is dispensable for the ATM-Parkin interaction. Both KU60019 and kinase-dead ATM (KD-ATM) complex with Parkin (or GFPParkin) in multiple cancer cell lines. This novel ATM-kinaseindependent affinity to complex with Parkin was further supported by the observation that ATM protein but not its kinase activity is required for mitophagy. Our data are in contrast with those of spermidine-induced mitophagy through ATM kinase-dependent activation of the PINK1/Parkin pathway in A-T fibroblasts.49 Moreover, recent studies provide evidence of the presence of mitochondrial Parkin23 in untreated A-T fibroblasts suggesting that ATM may not be required for mitochondrial Parkin translocation. However, we show that FCCP triggered both PINK1 and Parkin accumulation, consistent with spermidine- induced mitophagy.49 A recent study demonstrated that KU60019 treatment alleviates senescence via restoration, functional recovery and re-acidification of the lysosome/ autophagy system and metabolic reprogramming.50 Therefore, ATM kinase negatively regulates terminal mitochondrial destruction via strict control of lysosomal pH, underscoring our observation.
Our data also contradict the role of ATM kinase in mitophagy in multiple cancer cell lines and in primary lymphomas. Consistent with data from shATM cell lines and given that upstream Pink1 kinase activates and recruits Parkin into depolarized mitochondria, we also present evidence that ATM kinase activity is not required for either Parkin-UBSer65 phosphorylation or upstream Pink1 activation since neither inhibition of ATM kinase by KU60019 nor activation of kinase activity by neocarzinostatin affected the ATM-Parkin interaction or Parkin- UBSer65 phosphorylation. Similarly, despite heterogeneity among primary B-cell lymphomas, we did not see evidence of ATM kinase dependency in mitophagy. Consistent with the cell line data, we showed that ATM is not required for loss of m, and mitophagy is not controlled by the MCL-specific t(11;14) translocation in primary lymphomas. Although we do not know the 11q status of these lymphomas, a few primary MCL do not express detectable ATM in immunoblots and also have low or undetectable Parkin expression. Based on our IRinduced kinase screening assay, we predict several lymphomas may have lost ATM protein and a subset of these lymphomas lack either Pink1 or Parkin proteins. Moreover, a few ATM kinase deficient (IR¯) lymphomas also activated FCCP-induced Parkin-UBSer65 phosphorylation suggesting that ATM kinase activity is dispensable for Parkin activation. It is likely that while Parkin (PARK2) is frequently deleted in human cancers and associated with CCND1 overexpression42 may also contribute to cell proliferation in MCL and confers resistance to mitophagy.
In conclusion, loss of ATM protein may not only contribute to genotoxic stress through ROS production but can also provoke adverse effects including loss of Parkin, preservation of defective mitochondria, inhibition of mitophagy and low mitochondrial respiration, all of which may contribute to refractory diseases. We propose a pathway in which ATM but not its kinase activity is associated with induction of mitophagy through an ATM-Parkin interaction in cancer cells underscoring the first described molecular role of ATM in mitochondrial autophagy. Pharmacological targeting of mitophagy through ATM and Parkin, in combination with other drugs or stresses, may offer opportunities to control tumor progression because of the acute sensitivity of tumor cells to mitochondrial dysfunction.
- Received August 9, 2019
- Accepted January 31, 2020
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