Page 196 - 2021_02-Haematologica-web

A. Sarkar et al.
NCS (n=5 in Jeko-1 and n=3 in Mino) were done. Data represent the mean ± SEM; *P<0.05, **P<0.01, ***P<0.001: significant differences from respective con- trols. (G) Immunoblot analysis (30 μg total protein) of WT HeLa cells treated with FCCP, KU60019 or their combination, as in (E), and probed with the indicated anti- bodies. NCS served as a positive control for both ATMSer1981 and Kap1Ser824 activation. Total and phosphorylated bands were merged and are shown in color for speci- ficity. Separate blots were cut into pieces and probed with the indicated antibodies. The levels of Parkin, phospho-UBSer65 Parkin and Pink1 protein expression were detected by ECL. GAPDH was probed as a loading control each time. (H) Densitometry analysis of mitophagy (Tom20 expression) in WT HeLa cells treated with the indicated compounds, as in (F). Assays with FCCP, KU60019 or both in combination (n=6) and NCS (n=4) were performed. Data represent the mean ± SEM; *P<0.05: significant difference from the respective control. (I) Representative FCS analysis of irradiation (IR)-induced (5 Gray as in Figure 1G) ATMSer1981 and +H2AXSer139 phos- phorylation in B cells isolated from healthy donors (upper panel) or FCCP (75 mM for 3 h)-induced mitophagy (as in Figure 1A; lower panel). (J) Representative FCS analysis showing IR¯ and IR+ primary MCL lymphomas proficient or deficient in FCCP-induced mitophagy, respectively. (K) Line graph representation of the geometric mean of FCS analysis of mitochondrial retention in samples from four healthy donors (purified B cells in 3 cases and peripheral blood mononuclear cells in 1 case) or from 21 patients with primary MCL showing their individual IR status (IR+ n=13; IR– n=8) and their response to FCCP-induced mitophagy. (L) Quantitative poly- merase chain reaction (qRT-PCR) analysis (as in Figure 2I) of mitochondrial DNA (mtDNA) copy number in 20 primary MCL (IR+ n=12 and IR– n=8) or control healthy donor B cells (n=3). WT DNA and Rho0-DNA served as positive and negative controls for mtDNA qRT-PCR analysis. Inset showing mean ± SEM; *P<0.05 significant difference from IR+ lymphomas. (M) Relative geometric mean of basal mitochondrial reactive oxygen species (mROS) levels (as in Figure 1D) in healthy donor B cells
(n=3) or primary MCL (IR+ n=9 and IR– n=5). (N) Line graph representation of the geometric mean of FCS analysis of mitochondrial membrane potential (DΨ
ing FCCP treatment of cells from four healthy donors (3 purified B cells in 3 cases and peripheral blood mononuclear cells in 1 case) and from 21 patients with pri-
mary MCL showing their IR status (IR+ n=13; IR– n=8) and their response to FCCP-induced loss of DΨ
DMSO controls. (O) FCS analysis showing geometric means of mitochondrial mass following FCCP-induced mitophagy in cells from 21 patients with primary MCL and their respective t(11;14) status (positive n=18; negative n=3). (P) Immunoblot analysis showing FCCP-induced mitophagy in control B cells, MCL cell lines or six pri- mary MCL cases (30 μg total protein), probed with the indicated antibodies. Corresponding IR- or FCCP-induced FCS analysis data are shown in the tables under- neath. The levels of Parkin, phospho-UBSer65 Parkin and Pink1 protein expression were determined by an ECL method. Both actin and GAPDH were probed as loading controls. D: DMSO; F: FCCP; I: IR. (Q) Densitometry analysis of Parkin expression in purified B cells from healthy donors (n=3) or IR+ (n=22) and IR– (n=8) lymphomas (including MCL, marginal zone lymphoma, diffuse large B-cell lymphoma and follicular lymphoma). Data represent mean ± SEM.
. *P<0.05, **P<0.01: significant differences from respective m
tion, functional recovery and re-acidification of the lyso- some/autophagy system and metabolic reprogramming.50 Therefore, ATM kinase negatively regulates terminal mito- chondrial 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 neocarzi- nostatin affected the ATM-Parkin interaction or Parkin- UBSer65 phosphorylation. Similarly, despite heterogeneity among primary B-cell lymphomas, we did not see evi- dence of ATM kinase dependency in mitophagy. Consistent with the cell line data, we showed that ATM
) follow- m
observations, we propose that ATM kinase activity is dis- pensable for Parkin activation. Moreover, we observed that mitophagy inhibition following loss of ATM pro- motes high mROS and mtDNA copy number, low mito- chondrial 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 demon- strated 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 sup- port 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 stabili- ty of Parkin in tumor and non-tumor cells may enforce dif- ferent 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 dispen- sable for the ATM-Parkin interaction. Both KU60019 and kinase-dead ATM (KD-ATM) complex with Parkin (or GFP- Parkin) in multiple cancer cell lines. This novel ATM-kinase- independent 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 mito- chondrial 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 spermi- dine-induced mitophagy.49 A recent study demonstrated that KU60019 treatment alleviates senescence via restora-
is not required for loss of DΨ m
, and mitophagy is not con- trolled by the MCL-specific t(11;14) translocation in pri- mary lymphomas. Although we do not know the 11q sta- tus 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 IR- induced kinase screening assay, we predict several lym- phomas 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 phosphoryla- tion 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 con- tribute 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
510
haematologica | 2021; 106(2)