Eric Weterings

The endless tale of non-homologous end-joining

DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and non-homologous end-joining (NHEJ). The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.

Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

Ku recruits XLF to DNA double‐strand breaks

XRCC4‐like factor (XLF)—also known as Cernunnos—has recently been shown to be involved in non‐homologous end‐joining (NHEJ), which is the main pathway for the repair of DNA double‐strand breaks (DSBs) in mammalian cells. XLF is likely to enhance NHEJ by stimulating XRCC4–ligase IV‐mediated joining of DSBs. Here, we report mechanistic details of XLF recruitment to DSBs. Live cell imaging combined with laser micro‐irradiation showed that XLF is an early responder to DSBs and that Ku is essential for XLF recruitment to DSBs. Biochemical analysis showed that Ku–XLF interaction occurs on DNA and that Ku stimulates XLF binding to DNA. Unexpectedly, XRCC4 is dispensable for XLF recruitment to DSBs, although photobleaching analysis showed that XRCC4 stabilizes the binding of XLF to DSBs. Our observations showed the direct involvement of XLF in the dynamic assembly of the NHEJ machinery and provide mechanistic insights into DSB recognition.

DNA-dependent protein kinase in nonhomologous end joining: a lock with multiple keys?

The DNA-dependent protein kinase (DNA-PK) is one of the central enzymes involved in DNA double-strand break (DSB) repair. It facilitates proper alignment of the two ends of the broken DNA molecule and coordinates access of other factors to the repair complex. We discuss the latest findings on DNA-PK phosphorylation and offer a working model for the regulation of DNA-PK during DSB repair.

The Ku80 Carboxy Terminus Stimulates Joining and Artemis-Mediated Processing of DNA Ends

ABSTRACT Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and the XRCC4/ligase IV complex. Activation of the DNA-PKCS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PKCS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PKCS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PKCS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.

A novel small molecule inhibitor of the DNA repair protein Ku70/80.

Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.

Disruption of Aneuploidy and Senescence Induced by Aurora Inhibition Promotes Intrinsic Apoptosis in Double Hit or Double Expressor Diffuse Large B-cell Lymphomas

Double hit (DH) or double expressor (DE) diffuse large B-cell lymphomas (DLBCL) are aggressive non-Hodgkins lymphomas (NHL) with translocations and/or overexpressions of MYC and BCL-2, which are difficult to treat. Aurora kinase (AK) inhibition with alisertib in DH/DE-DLBCL induces cell death in ∼30%, while ∼70% are aneuploid and senescent cells (AASC), a mitotic escape mechanism contributing to drug resistance. These AASCs elaborated a high metabolic rate by increased AKT/mTOR and ERK/MAPK activity via BTK signaling through the chronic active B-cell receptor (BCR) pathway. Combinations of alisertib + ibrutinib or alisertib + ibrutinib + rituximab significantly reduced AASCs with enhanced intrinsic cell death. Inhibition of AK + BTK reduced phosphorylation of AKT/mTOR and ERK-1/2, upregulated phospho-H2A-X and Chk-2 (DNA damage), reduced Bcl-6, and decreased Bcl-2 and Bcl-xL and induced apoptosis by PARP cleavage. In a DE-DLBCL SCID mouse xenograft model, ibrutinib alone was inactive, while alisertib + ibrutinib was additive with a tumor growth inhibition (TGI) rate of ∼25%. However, TGI for ibrutinib + rituximab was ∼50% to 60%. In contrast, triple therapy showed a TGI rate of >90%. Kaplan–Meier survival analysis showed that 67% of mice were alive at day 89 with triple therapy versus 20% with ibrutinib + rituximab. All treatments were well tolerated with no changes in body weights. A novel triple therapy consisting of alisertib + ibrutinib + rituximab inhibits AASCs induced by AK inhibition in DH/DE-DLBCL leading to a significant antiproliferative signal, enhanced intrinsic apoptosis and may be of therapeutic potential in these lymphomas. Mol Cancer Ther; 16(10); 2083–93. ©2017 AACR.

Co-targeting aurora kinase with PD-L1 and PI3K abrogates immune checkpoint mediated proliferation in peripheral T-cell lymphoma: a novel therapeutic strategy

Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate ∼30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4+/CD8+ cells, we hypothesized that Program Death Ligand-1 (PD-L1) expression is essential for uncontrolled proliferation. Combination of alisertib with PI3Kα (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (∼9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-κB expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) ∼30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI >90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.

Abstract 2749: A novel small molecule inhibitor targets the DNA double strand break repair protein Ku70/80

Purpose: Non-Homologous End-Joining (NHEJ) is the primary pathway for the repair of DNA double strand breaks (DSBs) in human and mammalian cells. NHEJ is frequently upregulated in diverse solid malignancies and is a determining factor in the process of carcinogenesis. In addition, upregulation of the NHEJ pathway can reduce the efficiency of anti-cancer therapies including radiation or chemotherapy. Inhibition of NHEJ by siRNA reduces the growth of NHEJ-upregulated tumors and alleviates radiation-refractory disease. The Ku70/80 heterodimer is the central scaffolding protein of the NHEJ pathway, which recruits and activates all other key NHEJ factors. Therefore, Ku70/80 protein is a logical target for disruption of the NHEJ process. Experimental Procedures: We report the identification of a prospective binding pocket in the Ku70/80 crystal structure, located in close proximity to the DNA-binding domain and the Ku70/80 heterodimer interface. Based on this pocket, an idealized virtual ligand (‘protomol’) was created and utilized to screen ‘in silico’ an extensive database of small molecule compounds for potential ligands. Nine of the highest ranking hits were chosen for biological evaluation. Results: We identified one compound (designated ‘Compound L’), which proved capable of disrupting the in vitro binding of Ku70/80 to a DNA substrate in a dose-dependent manner with an IC50 of 3.5 μM. In addition, Compound L showed dose-dependent inhibition of DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKCS) activity, which requires Ku70/80 as a co-factor with an IC50 of 2.5 μM. Finally, we showed that Compound L synergistically sensitizes a glioblastoma cell line to ionizing radiation, with a marked sensitization at ∼20-25 μM. This latter observation clearly demonstrates the ability of Compound L to disrupt NHEJ-mediated repair of radiation-induced DSBs. Conclusions: Compound L is an active inhibitor of the Ku70/80 heterodimer. To the best of our knowledge, no such compound has been reported in the peer-reviewed literature. The structure of Compound L will be utilized as a scaffold for a ‘structure-activity relationship’ (SAR) based hit-to-lead drug development aimed at designing a novel class of anti-cancer agents targeting the Ku70/80 heterodimer. We predict Ku70/80 inhibitors would have applicability as single-modality drugs, following a synthetic lethality approach, or as sensitizing agents for concurrent radiation or chemotherapy in a variety of solid malignancies. Support: P30 CA023074. Citation Format: Daruka Mahadevan, Alfred C. Gallegos, Lauren N. Dominick, Laurence S. Cooke, Trace N. Bartels, Josef Vagner, Terry O. Matsunaga, Eric Weterings. A novel small molecule inhibitor targets the DNA double strand break repair protein Ku70/80. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2749.

Nonhomologous End Joining in Eukaryotes

This article discusses the core mechanism of the nonhomologous end-joining (NHEJ) process: one of two pathways that mediate the repair of DNA double-strand breaks (DSBs) in eukaryotic cells. Through complex interplay of several enzymes, NHEJ facilitates the capture, tethering, processing, and ligation of the two ends of a broken DNA molecule. The individual roles of all key NHEJ enzymes are discussed, as well as the rare human disease patterns that can arise by impairment of these enzymes. In addition, a brief description is given of cellular processes, other than DSB repair, that also utilize the NHEJ machinery.