Phosphorylation of Rad9 at Serine 328 by Cyclin A-Cdk2 Triggers Apoptosis via Interfering Bcl-xL

Zhuo, Zhan; He, Kan; Zhu, Dan; Jiang, Dan; Huang, Yinghui; Yang, Li; Sun, Chao

doi:10.1371/journal.pone.0044923

Cited by 12 publications

(8 citation statements)

References 36 publications

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“…Increased levels of Bax directly promote apoptosis, while Noxa and PUMA inhibit multiple anti-apoptotic BCL-2 proteins including BCL-XL and MCL-1, important because BCL-XL and MCL-1 overexpression is implicated in venetoclax resistance (37,38). In a second pathway, DNA damage acts independently of p53 by activating the checkpoint protein RAD9, which inhibits anti-apoptotic BCL-XL (39). Hence, RT both directly promotes apoptosis and inhibits anti-apoptotic alternatives to BCL-2, thus complementing the selective BCL-2 inhibition of venetoclax (4,12,40) and suggesting that combining RT with venetoclax may be valuable even in venetoclax-resistant disease.…”

Section: Discussionmentioning

confidence: 99%

Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas

et al. 2017

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Constitutive B-cell receptor signaling leads to overexpression of the anti-apoptotic BCL-2 protein and is implicated in the pathogenesis of many types of B-cell Non-Hodgkin Lymphoma (B-NHL). The BCL-2 small molecule inhibitor venetoclax shows promising clinical response rates in several lymphomas, but is not curative as monotherapy. Radiotherapy (RT) is a rational candidate for combining with BCL-2 inhibition, as DNA damage caused by RT increases the activity of pro-apoptotic BCL-2 pathway proteins, and lymphomas are exquisitely sensitive to radiation. We tested B-NHL responses to venetoclax combined with either external beam RT or radioimmunotherapy (RIT), which joins the selectivity of antibody targeting with the effectiveness of irradiation. We first tested cytotoxicity of cesium-137 irradiation plus venetoclax in 14 B-NHL cell lines representing five lymphoma sub-types. Combination treatment synergistically increased cell death in ten of 14 lines. Lack of synergy was predicted by resistance to single-agent venetoclax and high BCL-XL expression. We then assessed the efficacy of external beam RT plus venetoclax in murine xenograft models of mantle cell (MCL), germinal-center diffuse large B-cell (GCB-DLBCL), and activated B-cell (ABC-DLBCL) lymphomas. In each model, external beam RT plus venetoclax synergistically increased mouse survival time, curing up to 10%. We finally combined venetoclax treatment of MCL and ABC-DLBCL xenografts with a pretargeted RIT (PRIT) system directed against the CD20 antigen. Optimal dosing of PRIT plus venetoclax cured 100% of mice with no detectable toxicity. Venetoclax combined with RT may be a promising treatment for a wide range of lymphomas.

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Section: Discussionmentioning

confidence: 99%

Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas

et al. 2017

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“…This region is not required for 9-1-1 complex formation [ 11 , 28 ] but it is necessary for TopBP1 association and proper checkpoint function [ 15 , 17 , 29 ]. In addition, it is heavily modified by phosphorylation both constitutively and transiently in response to cell cycle position and DNA damage, and hence represents a potential regulatory mechanism for checkpoint control [ 15 , 30 – 32 ]. For example, S272 is phosphorylated rapidly and transiently in response to damage regardless of cell cycle position by the PI3K-related kinase ataxia telangiactasia-mutated (ATM) [ 15 , 33 ], T292 is targeted during mitosis by Cdc2 [ 30 ], and S341 and S387 are both phosphorylated constitutively and are required for the interaction between Rad9 and TopBP1 [ 15 , 31 ].…”

Section: Introductionmentioning

confidence: 99%

Tousled-Like Kinase-Dependent Phosphorylation of Rad9 Plays a Role in Cell Cycle Progression and G2/M Checkpoint Exit

Kelly

Davey

2013

PLoS ONE

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Genomic integrity is preserved by checkpoints, which act to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex is a PCNA-like clamp that is loaded onto DNA at structures resulting from damage and is important for initiating and maintaining the checkpoint response. Rad9 possesses a C-terminal tail that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1) as a kinase that may modify Rad9. Here we show that Rad9 is phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. We also show that TLK1 and Rad9 interact constitutively, and that this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, we demonstrate via siRNA-mediated depletion that TLK1 is required for progression through S-phase in normally cycling cells, and that cells lacking TLK1 display a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 has previously been shown to be transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, we propose that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn serves to regulate the DNA damage response.

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“…Indeed, apoptosis is an ATP-consuming phenomenon, which is accompanied with an amplification in both primary or intrinsic (caspase-9) and executioner or extrinsic (caspase-3/7) caspases (eg, 10-fold and 2-fold increases in caspase-3/7 and caspase-9 activity, respectively). 42 As such, in majority of cells, it has been shown that upon apoptosis induction, content of cellular ATP hugely decreases, though the extent of the decrease varies (20%-40%) and largely depends on the cell type, type, and concentration of apoptosis inducer. 43 In conclusion, the results indicated that a biologically relevant concentration of AFB 1 (10 ng/mL) induces apoptosis in mammalian neutrophils, lymphocytes, and monocytes through ATP depletion and caspases activation.…”

Section: Discussionmentioning

confidence: 99%

Naturally Occurring Level of Aflatoxin B₁ Injures Human, Canine and Bovine Leukocytes Through ATP Depletion and Caspase Activation

et al. 2019

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Aflatoxin (AF) B1 is a potent hepatotoxic, mutagenic, teratogenic mycotoxin and may cause immune suppression/dysregulation in humans and animals. Toxic effects of AFB1 on key mammalian immune cells (ie, leukocytes) needs to be mechanistically elucidated. In this study, along with the determination of AFB1’s LC50 for certain leukocytes, we analyzed the effect of naturally occurring levels of AFB1 on apoptosis/necrosis of neutrophils, lymphocytes, and monocytes from healthy young humans (20- to 25-year-old male), dogs (1- to 2-year-old Persian/herd breed), and cattle (1- to 2-year-old cattle). Leukocytes were incubated for approximately 24 hours with naturally occurring levels of AFB1 (10 ng/mL). Intracellular adenosine triphosphate (ATP) depletion and caspase-3/7 activity were then determined by luciferase-dependent bioluminescence (BL). Furthermore, the necrotic leukocytes were measured using propidium iodide (PI)-related flow cytometry. A significant decrease (24%-45%, 33.2% ± 2.7%) in intracellular ATP content was observed in AFB1-treated neutrophils, lymphocytes, and monocytes in all studied mammals. Also, with such a low level (10 ng/mL) of AFB1, BL-based caspase-3/7 activity (BL intensity) in all 3 tested mammalian leukocyte lineages was noticeably increased (∼>2-fold). Flow cytometry-based PI staining (for viability assay) of the AFB1-treated leukocytes showed slightly/insignificantly more increase of necrotic (PI+) neutrophils, lymphocytes, and monocytes in human, dogs, and cattle. Even though in vitro LC50s for AFB1’ (∼20,000-40,000 ng/mL) were approximately 2,000 to 4,000 times higher than background, these studies demonstrate leukocytes from human and farm/companion animals are sensitive to naturally occurring levels of AFB1. The observed in vitro ATP depletion and caspase activation in AFB1-exposed leukocytes can partially explain the underlying mechanisms of AFB1-induced immune disorders in mammals.

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Phosphorylation of Rad9 at Serine 328 by Cyclin A-Cdk2 Triggers Apoptosis via Interfering Bcl-xL

Cited by 12 publications

References 36 publications

Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas

Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas

Tousled-Like Kinase-Dependent Phosphorylation of Rad9 Plays a Role in Cell Cycle Progression and G2/M Checkpoint Exit

Naturally Occurring Level of Aflatoxin B₁ Injures Human, Canine and Bovine Leukocytes Through ATP Depletion and Caspase Activation

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Phosphorylation of Rad9 at Serine 328 by Cyclin A-Cdk2 Triggers Apoptosis via Interfering Bcl-xL

Cited by 12 publications

References 36 publications

Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas

Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas

Tousled-Like Kinase-Dependent Phosphorylation of Rad9 Plays a Role in Cell Cycle Progression and G2/M Checkpoint Exit

Naturally Occurring Level of Aflatoxin B1 Injures Human, Canine and Bovine Leukocytes Through ATP Depletion and Caspase Activation

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Naturally Occurring Level of Aflatoxin B₁ Injures Human, Canine and Bovine Leukocytes Through ATP Depletion and Caspase Activation