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A. Sarkar et al.
pieces and probed with the indicated antibodies. GAPDH served as a loading control. (G) Endogenous co-immunoprecipitation of the Mino MCL cell line (500 mg for each treatment) either with rabbit anti-Parkin antibody and probed with mouse anti-ATM (upper lanes) or with rabbit anti-ATM antibody and probed with mouse anti- Parkin antibody (lower lanes, both ECL blots). Cells were pretreated with FCCP (50 mM for 3 h), KU60019 (10 mM for 1 h) or NCS (40 nM for 1 h) before IP. (H) Input controls (5%) of the IP analysis from Figure 5G. Immunoblot analysis showing FCCP-induced ATMSer1981 phosphorylation or inhibition by KU60019. NCS served as a positive control for both ATMSer1981 and Kap1Ser824 phosphorylation. Total and phosphorylated bands were merged and are shown in color for specificity. The Parkin blot was probed using ECL reagents. GAPDH was used as a loading control each time. (I) IP analysis of an endogenous ATM-Parkin interaction in total, cytoplasm, mito- chondria or trypsin-digested mitochondria in untreated Mino cells (10x106 cells in each group) following cell fractionation. The Parkin-ATM interaction was detected in total, cytoplasm or in the untreated mitochondria but not in isolated mitochondria treated with trypsin. Isolated cellular fractions were immunoprecipitated with rabbit-Parkin antibody and probed with mouse ATM antibody. A 10 mL input control from the total cell extract was loaded to specify the ATM band. Rabbit IgG served as a negative control. (J) Input controls (5%) of the IP analysis from (I). Immunoblot analysis showing presence of ATM in total, cytoplasm, or in undigested mitochon- dria but not in isolated mitochondria treated with trypsin. GAPDH and HSP90 served as positive controls for their cytoplasmic abundance. Tom20 served as an outer mitochondrial membrane translocase while TIM23 represents an inner membrane translocase in trypsin-digested mitochondria.
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 lym- phomas which were resistant to mitophagy. Similarly despite a lack of IR-induced ATM kinase activation, a sub- set 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¯ lym- phomas (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 phos- phorylation 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 sta- tistical correlation was observed between IR status, mROS
pho-UBSer65 Parkin activation was not detected in a few lymphomas (DLBCL 4, 5 and FL 2) rendering them resist- ant to mitophagy.
Discussion
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 thy- mocytes 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 possi- ble link between Parkin and ATM. Parkin is a tumor-sup- pressor 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 there- by 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 associat- ed with a high number of chromosomal alterations.43 MCL is an aggressive form of non-Hodgkin lymphoma, and remains largely incurable; most affected patients eventual- ly die of relapsed/refractory disease,44 thereby arguing for a need for an alternative method of treatment.
or loss of DΨ m
notion that Pink1 activation leading to Parkin activation Ser65
in these lymphomas, while mtDNA copy number (Online Supplementary Figure S9B-D) was higher in IR¯ FL and DLBCL. Although the majority of these lym- phomas expressed Pink1 and Parkin, FCCP-induced phos-
via UB phosphorylation during mitophagy is in part ATM-dependent. We provide evidence that KU60019 failed to inhibit FCCP-induced Parkin-UBSer65 phosphoryla- tion and mitophagy in multiple cell lines. Based on these
We present evidence that cancer cell mitophagy is dependent on ATM but not its kinase activity and con- nects 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
DΨ 45
ated mitophagy. Our data are in agreement with the
, a prerequisite signal required for Pink1-Parkin-medi- m
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haematologica | 2021; 106(2)