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DNA-PK inhibitor peposertib enhances p53-dependent cytotoxicity of DNA double-strand break inducing therapy in acute leukemia.

ABSTRACT: Peposertib (M3814) is a potent and selective DNA-PK inhibitor in early clinical development. It effectively blocks non-homologous end-joining repair of DNA double-strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By suppressing DNA-PK catalytic activity in the presence of DNA DSB, M3814 potentiates ATM/p53 signaling leading to enhanced p53-dependent antitumor activity in tumor cells. Here, we investigated the therapeutic potential of M3814 in combination with DSB-inducing agents in leukemia cells and a patient-derived tumor. We show that in the presence of IR or topoisomerase II inhibitors, M3814 boosts the ATM/p53 response in acute leukemia cells leading to the elevation of p53 protein levels as well as its transcriptional activity. M3814 synergistically sensitized p53 wild-type, but not p53-deficient, AML cells to killing by DSB-inducing agents via p53-dependent apoptosis involving both intrinsic and extrinsic effector pathways. The antileukemic effect was further potentiated by enhancing daunorubicin-induced myeloid cell differentiation. Further, combined with the fixed-ratio liposomal formulation of daunorubicin and cytarabine, CPX-351, M3814 enhanced the efficacy against leukemia cells in vitro and in vivo without increasing hematopoietic toxicity, suggesting that DNA-PK inhibition could offer a novel clinical strategy for harnessing the anticancer potential of p53 in AML therapy.

SUBMITTER: Haines E

PROVIDER: S-EPMC8190296 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Hyperactivation of DNA-PK by double-strand break mimicking molecules disorganizes DNA damage response.

Project description:Cellular response to DNA damage involves the coordinated activation of cell cycle checkpoints and DNA repair. The early steps of DNA damage recognition and signaling in mammalian cells are not yet fully understood. To investigate the regulation of the DNA damage response (DDR), we designed short and stabilized double stranded DNA molecules (Dbait) mimicking double-strand breaks. We compared the response induced by these molecules to the response induced by ionizing radiation. We show that stable 32-bp long Dbait, induce pan-nuclear phosphorylation of DDR components such as H2AX, Rpa32, Chk1, Chk2, Nbs1 and p53 in various cell lines. However, individual cell analyses reveal that differences exist in the cellular responses to Dbait compared to irradiation. Responses to Dbait: (i) are dependent only on DNA-PK kinase activity and not on ATM, (ii) result in a phosphorylation signal lasting several days and (iii) are distributed in the treated population in an "all-or-none" pattern, in a Dbait-concentration threshold dependant manner. Moreover, despite extensive phosphorylation of the DNA-PK downstream targets, Dbait treated cells continue to proliferate without showing cell cycle delay or apoptosis. Dbait treatment prior to irradiation impaired foci formation of Nbs1, 53BP1 and Rad51 at DNA damage sites and inhibited non-homologous end joining as well as homologous recombination. Together, our results suggest that the hyperactivation of DNA-PK is insufficient for complete execution of the DDR but induces a "false" DNA damage signaling that disorganizes the DNA repair system.

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Interaction of the double-strand break repair kinase DNA-PK and estrogen receptor-alpha.

Project description:Estrogens are suggested to play a role in the development and progression of proliferative diseases such as breast cancer. Like other steroid hormone receptors, the estrogen receptor-alpha (ERalpha) is a substrate of protein kinases, and phosphorylation has profound effects on its function and activity. Given the importance of DNA-dependent protein kinase (DNA-PK) for DNA repair, cell cycle progression, and survival, we hypothesized that it modulates ERalpha signaling. Here we show that, upon estrogen stimulation, DNA-PK forms a complex with ERalpha in a breast cancer cell line (MELN). DNA-PK phosphorylates ERalpha at Ser-118. Phosphorylation resulted in stabilization of ERalpha protein as inhibition of DNA-PK resulted in its proteasomal degradation. Activation of DNA-PK by double-strand breaks or its inhibition by siRNA technology demonstrated that estrogen-induced ERalpha activation and cell cycle progression is, at least, partially dependent on DNA-PK.

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G9a inhibition potentiates the anti-tumour activity of DNA double-strand break inducing agents by impairing DNA repair independent of p53 status.

Project description:Cancer cells often exhibit altered epigenetic signatures that can misregulate genes involved in processes such as transcription, proliferation, apoptosis and DNA repair. As regulation of chromatin structure is crucial for DNA repair processes, and both DNA repair and epigenetic controls are deregulated in many cancers, we speculated that simultaneously targeting both might provide new opportunities for cancer therapy. Here, we describe a focused screen that profiled small-molecule inhibitors targeting epigenetic regulators in combination with DNA double-strand break (DSB) inducing agents. We identify UNC0638, a catalytic inhibitor of histone lysine N-methyl-transferase G9a, as hypersensitising tumour cells to low doses of DSB-inducing agents without affecting the growth of the non-tumorigenic cells tested. Similar effects are also observed with another, structurally distinct, G9a inhibitor A-366. We also show that small-molecule inhibition of G9a or siRNA-mediated G9a depletion induces tumour cell death under low DNA damage conditions by impairing DSB repair in a p53 independent manner. Furthermore, we establish that G9a promotes DNA non-homologous end-joining in response to DSB-inducing genotoxic stress. This study thus highlights the potential for using G9a inhibitors as anti-cancer therapeutic agents in combination with DSB-inducing chemotherapeutic drugs such as etoposide.

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p53 binding during the DNA double-strand break response

Project description:We used ChIP-seq to assess where p53 binds in the human genome and how that binding changes during the DNA double-strand break response. In particular, we considered the 1-Mb-wide window centered on the MYC locus. Contrary to previous reports, we found no evidence of p53 binding at the MYC promoter. Rather, we identified three locations downstream of MYC at which p53 was bound; binding at each of these regions increased during the DNA double-strand break response.

2017-08-02 | GSE101737 | GEO

Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair.

Project description:p53-binding protein 1 (53BP1) exerts distinct impacts in different situations involving DNA double-strand break (DSB) rejoining. Here we focus on how 53BP1 impacts upon the repair of ionising radiation-induced DSBs (IR-DSBs) and how it interfaces with Ku, the DNA end-binding component of canonical non-homologous end-joining (c-NHEJ), the major DSB repair pathway in mammalian cells. We delineate three forms of IR-DSB repair: resection-independent c-NHEJ, which rejoins most IR-DSBs with fast kinetics in G1 and G2, and Artemis and resection-dependent c-NHEJ and homologous recombination (HR), which repair IR-DSBs with slow kinetics in G1 and G2 phase, respectively. The fast component of DSB repair after X-ray exposure occurs via c-NHEJ with normal kinetics in the absence of 53BP1. Ku is highly abundant and has avid DNA end-binding capacity which restricts DNA end-resection and promotes resection-independent c-NHEJ at most IR-DSBs. Thus, 53BP1 is largely dispensable for resection-independent c-NHEJ. In contrast, 53BP1 is essential for the process of rejoining IR-DSBs with slow kinetics. This role requires 53BP1's breast cancer susceptibility gene I (BRCA1) C-terminal (BRCT) 2 domain, persistent ataxia telangiectasia mutated (ATM) activation and potentially relaxation of compacted chromatin at heterochromatic-DSBs. In distinction, 53BP1 inhibits resection-dependent IR-DSB repair in G1 and G2, and this resection-inhibitory function can be counteracted by BRCA1. We discuss a model whereby most IR-DSBs are rapidly repaired by 53BP1-independent and resection-independent c-NHEJ due to the ability of Ku to inhibit resection, but, if delayed, then resection in the presence of Ku is triggered, the 53BP1 barrier comes into force and BRCA1 counteraction is required for resection.

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DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining.

Project description:DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.

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LRRK2 inhibition potentiates PARP inhibitor cytotoxicity through inhibiting homologous recombination-mediated DNA double strand break repair.

Project description:PARP inhibitors induce DNA lesions, the repair of which are highly dependent on homologous recombination (HR), and preferentially kill HR- deficient cancers. However, cancer cells have developed several mechanisms to transform HR and confer drug resistance to PARP inhibition. Therefore, there is a great clinical interest in exploring new therapies that induce HR deficiency (HRD), thereby sensitizing cancer cells to PARP inhibitors. Here, we found that GSK2578215A, a high-selective and effective leucine-rich repeat kinase 2 (LRRK2) inhibitor, or LRRK2 depletion suppresses HR preventing the recruitment of RAD51 to DNA damage sites through disruption of the interaction of RAD51 and BRCA2. Moreover, LRRK2 inhibition or depletion increases the susceptibility of ovarian cancer cells to Olaparib in vitro and in vivo. In clinical specimens, LRRK2 high expression is high related with advanced clinical characteristics and poor survival of ovarian cancer patients. All these findings indicate ovarian cancers expressing high levels of LRRK2 are more resistant to treatment potentially through promoting HR. Furthermore, combination treatment with an LRRK2 and PARP inhibitor may be a novel strategy to improve the effectiveness of LRRK2 expression ovarian cancers.

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Barrier-to-autointegration-factor (Banf1) modulates DNA double-strand break repair pathway choice via regulation of DNA-dependent kinase (DNA-PK) activity.

Project description:DNA repair pathways are essential to maintain the integrity of the genome and prevent cell death and tumourigenesis. Here, we show that the Barrier-to-Autointegration Factor (Banf1) protein has a role in the repair of DNA double-strand breaks. Banf1 is characterized as a nuclear envelope protein and mutations in Banf1 are associated with the severe premature aging syndrome, Néstor-Guillermo Progeria Syndrome. We have previously shown that Banf1 directly regulates the activity of PARP1 in the repair of oxidative DNA lesions. Here, we show that Banf1 also has a role in modulating DNA double-strand break repair through regulation of the DNA-dependent Protein Kinase catalytic subunit, DNA-PKcs. Specifically, we demonstrate that Banf1 relocalizes from the nuclear envelope to sites of DNA double-strand breaks. We also show that Banf1 can bind to and directly inhibit the activity of DNA-PKcs. Supporting this, cellular depletion of Banf1 leads to an increase in non-homologous end-joining and a decrease in homologous recombination, which our data suggest is likely due to unrestrained DNA-PKcs activity. Overall, this study identifies how Banf1 regulates double-strand break repair pathway choice by modulating DNA-PKcs activity to control genome stability within the cell.

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