Protein 53BP1 Research Articles

Abstract A39: Understanding poly-ADP-ribose polymerase (PARP) inhibitor resistance in BRCA2-deficient cells through dual CRISPR knockout and activation screens

Abstract Cells deficient in the DNA repair pathway homologous recombination (HR) show particular sensitivity to inhibitors of poly-ADP-ribose polymerases (PARPi). Based on this observation, several PARPi, such as olaparib, have been FDA approved for the treatment of BRCA2-mutated ovarian cancer. Indeed, clinical trials have demonstrated that use of these agents significantly improved progression-free survival (PFS), for example, from 5.5 months to 19 months in one cohort of ovarian cancer patients harboring BRCA1/2 mutations (SOLO2 trial). However, even in this trial, only 65% of patients who were predicted to be genetically susceptible to this treatment attained 12 months PFS. This indicates that sensitivity to PARPi is mediated by more than simply BRCA2 status. Moreover, investigations into mechanisms governing sensitivity and resistance to PARPi continue to further our understanding of basic DNA repair and replication pathways. To this end, we conducted dual CRISPR screens in BRCA2-deficient HeLa cells as an unbiased approach for identifying proteins whose loss or overexpression confers resistance to PARPi. First, over 19,000 genes were individually knocked out in BRCA2-deficient cells. Then, these cells were treated or not with olaparib. Sequencing and computational analysis were used to identify which genes were lost most often in cells surviving olaparib treatment, yielding hundreds of potential hits. Hits were tested in multiple cell lines using cellular viability, apoptosis, and clonogenic survival assays. Several of these hits were successfully validated, including the histone acetyltransferase TIP60 (KAT5) and the ubiquitin ligase HUWE1. Mechanistically, we show that the rescue of PARPi sensitivity caused by TIP60 depletion is dependent on the downstream proteins 53BP1 and REV7, proteins known to inhibit end resection, thus promoting NHEJ; this indicates that the role of TIP60 as a modulator of double-strand break repair pathway choice underlies this resistance. The finding that increased 53BP1 binding contributes to PARPi resistance in BRCA2-deficient cells is particularly interesting given that loss of 53BP1 has been shown to cause resistance to PARPi in BRCA1-deficient cells. This indicates that pathways that govern sensitivity to PARPi in BRCA1-deficient cells may have opposite effects on this phenotype in BRCA2-deficient cells. To further understand the networks governing PARPi resistance in BRCA2-deficient cells, we also completed a CRISPR activation screen using the same experimental design as the knockout screen. By combining the knockout and activation screens for the first time in this context, we are able to better construct overarching networks regulating PARPi sensitivity in BRCA2-deficient cells. We have identified many potential mediators of PARPi resistance in BRCA2-deficient cells by combining CRISPR-knockout and CRISPR-activation screens. Our work identifies TIP60 as a novel potential biomarker for PARPi response in BRCA2-deficient cells. Citation Format: Kristen E. Clements, Anastasia Hale, Nathanial J. Tolman, George-Lucian Moldovan. Understanding poly-ADP-ribose polymerase (PARP) inhibitor resistance in BRCA2-deficient cells through dual CRISPR knockout and activation screens [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A39.

TIRR: a potential front runner in HDR race-hypotheses and perspectives.

The majority of CRISPR-Cas9 methods for mutations correction are oriented on gene editing through homologous recombination that is normally restrained by non-homologous end joining (NHEJ). A recently identified protein TIRR can bind a 53BP1 protein, a key effector of NHEJ, and inhibit its recruitment to double-strand break loci. Several studies elucidated the molecular mechanisms of TIRR-53BP1 binding and established bidirectional role of TIRR in 53BP1 functions and stability. It was proved that overexpression of TIRR promotes the double-strand break repair through homologous recombination. All findings, which were described in the review, allow assuming TIRR as a suitable target for enhancing efficacy of genome editing through homology directed repair.

Nudix Hydrolase NUDT16 Regulates 53BP1 Protein by Reversing 53BP1 ADP-Ribosylation.

53BP1 controls two downstream subpathways, one mediated by PTIP and Artemis and the other by RIF1 and MAD2L2/Shieldin, to coordinate DNA repair pathway choices. However, the upstream regulator(s) of 53BP1 function in DNA repair remain unknown. We and others recently reported that TIRR associates with 53BP1 to stabilize it and prevents 53BP1 localization to DNA damage sites by blocking 53BP1 Tudor domain binding to H4K20me2 sites. Here, we report that the Nudix hydrolase NUDT16, a TIRR homolog, regulates 53BP1 stability. We identified a novel posttranslational modification of 53BP1 by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to 53BP1 polyubiquitination and degradation. In response to DNA damage, ADP-ribosylated 53BP1 increased significantly, resulting in its ubiquitination and degradation. These data suggest that NUDT16 plays a major role in controlling 53BP1 levels under both normal growth conditions and during DNA damage. Notably, overexpression of a NUDT16 catalytically inactive mutant blocked 53BP1 localization to double-strand breaks because (i) the mutant binding to TIRR increased after IR; (ii) the mutant enhanced 53BP1 Tudor domain binding to TIRR, and (iii) the mutant impaired the interaction of 53BP1 Tudor domain with H4K20me2. Moreover, NUDT16's catalytic hydrolase activity was required for 53BP1 de-ADP-ribosylation, 53BP1 protein stability, and its function in cell survival. In summary, we demonstrate that NUDT16 regulates 53BP1 stability and 53BP1 recruitment at double-strand breaks, providing yet another mechanism of 53BP1 regulation.Significance: This study provides a novel mechanism of 53BP1 regulation by demonstrating that NUDT16 has hydrolase activities that remove ADP-ribosylation of 53BP1 to regulate 53BP1 stability and 53BP1 localization at DSBs.

Reshaping Chromatin Architecture around DNA Breaks

DNA double-strand breaks (DSBs) elicit major chromatin changes. Using super-resolution microscopy in human cells, Ochs etal. unveil that the DSB response protein 53BP1 and its effector RIF1 organize DSB-flanking chromatin into circular micro-domains. These structures control the spatial distribution of DSB repair factors safeguarding genome integrity.

STAG2 Loss Gives Rise to Therapeutically Targetable DNA Damage Repair Defects and Altered Replication Fork Dynamics in Acute Myeloid Leukaemia

Acute Myeloid Leukaemia (AML) is a genetically heterogeneous disease of the blood and bone marrow primarily affecting older populations. Chemotherapy has been the standard of care for four decades, just 10% of elderly patients show a response and it will be curative in only a third of patients aged 18-60. Therefore, it's necessary to identify new therapeutic targets for improved treatment options. The search for novel mutations within the genomes of karyotypically normal AML patients identified Cohesin's STAG2 as a recurrently mutated gene (6%). Cohesin is a ring-shaped multimeric protein complex which has a multitude of roles including maintaining sister chromatid cohesion, DNA replication and repair, gene expression and chromatin architecture. STAG2 remains the most understudied Cohesin gene, how a mutation affects the ability of Cohesin to fulfil certain roles needs elucidation as well as how STAG2 functions as part of Cohesin to repair DNA breaks, promote faithful replication and control gene expression. Therefore, we sought to explore the consequences of loss-of-function STAG2 mutations on DNA repair and replication using a STAG2 knockout (ΔSTAG2) isogenic cell line model created using CRISPR/Cas9. Immunofluorescent staining of DNA damage repair proteins 53BP1 and RAD51 following 2Gy irradiation (IR) suggests ΔSTAG2 cells have a significant DNA repair defect. The average 53BP1 and RAD51 foci counts were significantly higher (p=0.001) in mutant cells which had three times as many foci at 24 hours after IR. In addition, 40% of ΔSTAG2 cells stain positive for 53BP1 and RAD51 respectively at 24 hours post-IR. This damage persists at 48 and 72 hours, whereas wild type (WT) damage returns to basal levels. Given that our data suggests a HR defect, we assessed the response of WT and ΔSTAG2 cells to PARP inhibitors (PARPi). Interestingly, ΔSTAG2 cells showed significantly greater sensitivity than WT cells to highly potent PARPi Talazoparib with an IC50 of 62nM. We next assayed single DNA fibres to evaluate the implications of a STAG2 mutation on the dynamics of individual replication forks. Interestingly, we observed a significantly reduced number of ongoing replication forks compared to WT cells (p=0.01) with a concomitant increase in the number of stalled forks (p=0.01) in the absence of exogeneous DNA damage. Following treatment with hydroxyurea (HU) this phenotype was exacerbated and ΔSTAG2 cells exhibit significantly more stalled replication forks than WTs (p=0.001). This indicates a role for STAG2 in maintaining replication fork stability and promoting fork restart following replication stress. Importantly, our data identifies a potential DNA damage repair deficiency that sensitises ΔSTAG2 cells to PARPi monotherapy as well as unearthing an essential role for STAG2 in replication fork progression and fork stability. This mutation could therefore be exploited therapeutically and represents a novel therapeutic target/approach for AML patients with mutated STAG2. Disclosures No relevant conflicts of interest to declare.

Oxidative DNA damage is concurrently repaired by base excision repair (BER) and apyrimidinic endonuclease 1 (APE1)-initiated nonhomologous end joining (NHEJ) in cortical neurons.

AimsAccumulating studies have suggested that base excision repair (BER) is the major repair pathway of oxidative DNA damage in neurons, and neurons are deficient in other DNA repair pathways, including nucleotide excision repair and homologous recombination repair. However, some studies have demonstrated that neurons could efficiently repair glutamate‐ and menadione‐induced double‐strand breaks (DSBs), suggesting that the DSB repair mechanisms might be implicated in neuronal health. In this study, we hypothesized that BER and nonhomologous end joining (NHEJ) work together to repair oxidative DNA damage in neurons.MethodsImmunohistochemistry and confocal microscopy were employed to examine the colocalization of apyrimidinic endonuclease 1 (APE1), histone variant 2AX (γH2AX) and phosphorylated p53‐binding protein (53BP1). APE1 inhibitor and shRNA were respectively applied to suppress APE1 activity and protein expression to determine the correlation of APE1 and DSB formation. The neutral comet assay was used to determine and quantitate the formation of DSB.ResultsBoth γH2AX and 53BP1 were upregulated and colocalized with APE1 in the nuclei of rat cortical neurons subjected to menadione‐induced oxidative insults. Phospho53BP1 foci were efficiently abolished, but γH2AX foci persisted following the suppression of APE1 activity. Comet assays demonstrated that the inhibition of APE1 decreased the DSB formation.ConclusionsOur results indicate that APE1 can engage the NHEJ mechanism in the repair of oxidative DNA damage in neurons. These findings provide insights into the mechanisms underlying the efficient repair of oxidative DNA damage in neurons despite the high oxidative burden.

Physiological and pathological roles of LRRK2 in the nuclear envelope integrity.

Mutations in LRRK2 cause autosomal dominant and sporadic Parkinson's disease, but the mechanisms involved in LRRK2 toxicity in PD are yet to be fully understood. We found that LRRK2 translocates to the nucleus by binding to seven in absentia homolog (SIAH-1), and in the nucleus it directly interacts with lamin A/C, independent of its kinase activity. LRRK2 knockdown caused nuclear lamina abnormalities and nuclear disruption. LRRK2 disease mutations mostly abolish the interaction with lamin A/C and, similar to LRRK2 knockdown, cause disorganization of lamin A/C and leakage of nuclear proteins. Dopaminergic neurons of LRRK2 G2019S transgenic and LRRK2 -/- mice display decreased circularity of the nuclear lamina and leakage of the nuclear protein 53BP1 to the cytosol. Dopaminergic nigral and cortical neurons of both LRRK2 G2019S and idiopathic PD patients exhibit abnormalities of the nuclear lamina. Our data indicate that LRRK2 plays an essential role in maintaining nuclear envelope integrity. Disruption of this function by disease mutations suggests a novel phosphorylation-independent loss-of-function mechanism that may synergize with other neurotoxic effects caused by LRRK2 mutations.

In vitro exposure to thiacloprid-based insecticide formulation promotes oxidative stress, apoptosis and genetic instability in bovine lymphocytes

A proprietary thiacloprid-based neonicotinoid insecticide formulation is widely used in agriculture to protect vegetables and fruit against various pests. However, its effect on animal cells has not been fully elucidated. In this study, bovine peripheral lymphocytes were incubated with different concentrations of this formulation (10; 30; 60; 120 and 240 μg.mL−1) for 4 h to address the potential cytotoxic and genotoxic effects of the insecticide. Insecticide formulation treatment resulted in decreased cell viability and proliferation, p53-mediated cell cycle arrest at the G0/G1 phase, and apoptosis induction accompanied by elevated levels of mitochondrial superoxide and protein carbonylation. Oxidant-based DNA damage and DNA damage response (DDR) were also observed, namely the formation of micronuclei, DNA double-strand breaks and slightly elevated recruitment of p53 binding protein (53BP1) foci. Our results contribute to the elucidation of insecticide effects on animal lymphocyte cultures after short-term exposure. Due to increased application of neonicotinoids worldwide, resulting in both higher yields and adverse effects on non-target animals and humans, further in vivo and in vitro experiments should be performed to confirm their cytotoxic and genotoxic activities during short-term exposure.

Beyond the Trinity of ATM, ATR, and DNA-PK: Multiple Kinases Shape the DNA Damage Response in Concert With RNA Metabolism.

Our genome is constantly exposed to endogenous and exogenous sources of DNA damage resulting in various alterations of the genetic code. DNA double-strand breaks (DSBs) are considered one of the most cytotoxic lesions. Several types of repair pathways act to repair DNA damage and maintain genome stability. In the canonical DNA damage response (DDR) DSBs are recognized by the sensing kinases Ataxia-telangiectasia mutated (ATM), Ataxia-telangiectasia and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK), which initiate a cascade of kinase-dependent amplification steps known as DSB signaling. Recent evidence suggests that efficient recognition and repair of DSBs relies on the transcription and processing of non-coding (nc)RNA molecules by RNA polymerase II (RNAPII) and the RNA interference (RNAi) factors Drosha and Dicer. Multiple kinases influence the phosphorylation status of both the RNAPII carboxy-terminal domain (CTD) and Dicer in order to regulate RNA-dependent DSBs repair. The importance of kinase signaling and RNA processing in the DDR is highlighted by the regulation of p53-binding protein (53BP1), a key regulator of DSB repair pathway choice between homologous recombination (HR) and non-homologous end joining (NHEJ). Additionally, emerging evidence suggests that RNA metabolic enzymes also play a role in the repair of other types of DNA damage, including the DDR to ultraviolet radiation (UVR). RNAi factors are also substrates for mitogen-activated protein kinase (MAPK) signaling and mediate the turnover of ncRNA during nucleotide excision repair (NER) in response to UVR. Here, we review kinase-dependent phosphorylation events on RNAPII, Drosha and Dicer, and 53BP1 that modulate the key steps of the DDR to DSBs and UVR, suggesting an intimate link between the DDR and RNA metabolism.

Effect of low-dose tungsten on human thyroid stem/precursor cells and their progeny.

Thyroid cancer incidence is increased in volcanic areas where environment pollution biocontaminates residents. Tungsten (W) is the most increased heavy metal in drinking water of Mount Etna volcanic area where it exceeds the normal range in the urine of 27% inhabitants. The possible connection between increased tungsten and thyroid cancer has never been studied. We investigated in vitro the effect tungsten on both human thyrocytes in primary culture, thyrospheres (aggregates of stem/precursor thyroid cells) and thyrocytes differentiated from tungsten-exposed thyrospheres. Chronic exposure to low-dose (nanomolar range, as in the urines of volcanic area residents) soluble tungsten had major biological effects on thyroid stem/precursor cells, promoting growth with a biphasic (hormetic) dose-response and reducing apoptosis. No such effects were observed in mature thyrocytes. In addition, tungsten-exposed thyrospheres had abnormal expression of genes commonly altered also in thyroid cancer and increased activation of the DNA-repair proteins H2AX and 53BP1. Moreover, exposure to tungsten decreased thyrosphere differentiation, as indicated by the reduced expression of thyroid-specific genes in derived thyrocytes that also showed preneoplastic changes such as increased anchorage-independent growth, clonogenic growth and migration capacity. The mechanism of action of tungsten on thyroid stem/precursor cells is unclear but involves membrane G-proteins and activation of the ERK signaling pathway. These data indicate that chronic exposure to slightly increased tungsten, harmless for mature thyrocytes, importantly affects the biology of stem/precursor thyroid cells and of their progeny, inducing characteristics of preneoplastic transformation.

Ultra-soft X-ray system for imaging the early cellular responses to X-ray induced DNA damage.

The majority of the proteins involved in processing of DNA double-strand breaks (DSBs) accumulate at the damage sites. Real-time imaging and analysis of these processes, triggered by the so-called microirradiation using UV lasers or heavy particle beams, yielded valuable insights into the underlying DSB repair mechanisms. To study the temporal organization of DSB repair responses triggered by a more clinically-relevant DNA damaging agent, we developed a system coined X-ray multi-microbeam microscope (XM3), capable of simultaneous high dose-rate (micro)irradiation of large numbers of cells with ultra-soft X-rays and imaging of the ensuing cellular responses. Using this setup, we analyzed the changes in real-time kinetics of MRE11, MDC1, RNF8, RNF168 and 53BP1—proteins involved in the signaling axis of mammalian DSB repair—in response to X-ray and UV laser-induced DNA damage, in non-cancerous and cancer cells and in the presence or absence of a photosensitizer. Our results reveal, for the first time, the kinetics of DSB signaling triggered by X-ray microirradiation and establish XM3 as a powerful platform for real-time analysis of cellular DSB repair responses.

Formation of DNA Double-Strand Breaks in Rat Brain Neurons after Irradiation with Krypton Ions (78Kr)

The induction and repair of DNA double-strand breaks in hippocampal formation after exposure to accelerated 78Kr ions with an energy of 2.58 GeV/nucleon has been studied using the immunohistochemical staining of rat-brain paraffin sections with antibodies specific to γH2AX and 53BP1 proteins. Following exposure to heavy ions, complex clusters of double-strand breaks are detected in various structures of hippocampal formation. It is shown that most γH2AX/53BP1 foci are part of complex clusters (~80%) and colocalized with visualized repair proteins (~75%) along the tracks of heavy charged particles. After 24 h of postirradiation incubation, the effective elimination of radiation-induced γH2AX/53BP1 foci is observed in the cells of the dentate gyrus and the CA1 region of the hippocampal formation. In the cells of the dentate gyrus, the peak of γH2AX/53BP1 formation occurs 4 h after irradiation.

Abstract 3797: AsiDNA abrogates acquired resistance to PARP inhibitors

Abstract Purpose: PARP inhibitors (PARPi) are approved for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance remain a clinical hurdle, and several attempts failed to decrease this resistance. In the current study, we propose a novel therapeutic strategy, based on drug combination to abolish the onset of resistance to PARPi. Experimental design: We performed a set of experiments to induce resistance to PARPi and assess the impact of AsiDNA addition to decrease this resistance occurrence. AsiDNA is a double stranded DNA molecule (decoy oligo-nucleotide) that mimics double stranded DNA breaks (DSBs) to interfere with DNA repair, by over-activating a “false” signaling of DNA damage through DNA-PK and PARP enzymes. We characterized the effects of long term treatment with the PARPi olaparib and talazoparib on resistance selection in two different cell lines, BC227 and NCI-H446, and we further analyzed the effect of low doses of AsiDNA on the occurrence of acquired resistance to these PARPi. Finally, we addressed the mechanisms underlying resistance emergence and abrogation. Results: Long term exposure of cancer cells to PARPi induces the emergence of resistance in all the tested independent populations (11 BC227 independent populations and 3 NCI-H446 for talazoparib, 5 BC227 independent populations for olaparib), raising the question of the clinical benefit of long-term maintenance monotherapy with PARPi. Interestingly, double-treated populations with AsiDNA (2.5µM - low non cytotoxic dose) and talazoparib or olaparib showed a significant lower probability of resistance occurrence. In fact, 9/11 (or 5/5) BC227 populations treated with AsiDNA and talazoparib (or AsiDNA and olaparib) remained sensitive up to 5 cycles of treatment, and 3/3 NCI-H446 populations treated with AsiDNA and talazoparib. Interestingly, AsiDNA is also able to partially revert talazoparib resistance in BC227 and NCI-H446 resistant populations. As described, PARP1, EZH2 and/or 53BP1 protein expression loss or reduction are important mechanisms leading to PARPi resistance. We confirmed in these experiments that treatments with PARPi significantly modify the expression and activity of these proteins and we demonstrated that co-treatment with AsiDNA inhibits these changes. These results indicate that AsiDNA may abrogate and reverse PARPi acquired resistance by the normalization of involved protein expression and activity. Conclusion: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate drug-driven PARPi resistance abrogation. As PARPi have obtained FDA approval, and AsiDNA is being currently tested in a clinical trial, a potential exists for a rapid clinical confirmation of AsiDNA-induced PARPi resistance abrogation and reversion. Citation Format: Wael Jdey, Pauline Lascaux, Françoise Bono. AsiDNA abrogates acquired resistance to PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3797.

Platforms for delivery of macromolecules to sites of DNA double-strand break repair

Double-strand break (DSB) repair foci are important therapeutic targets. Here we describe platforms for delivery of macromolecules, nanomaterials and nanomedicines to repair foci. The strategy is based on the high affinity of the human 53BP1 protein for modified chromatin present at sites of DNA damage. As proof of concept, we created, expressed, and purified an engineered fragment of 53BP1 and coupled it to fluorescent streptavidin, a model cargo with no intrinsic affinity for repair foci. This binary complex was in turn coupled to the iron carrier protein, transferrin, which engages a high-affinity cell surface receptor. In a different version of the complex, transferrin was omitted and a protein transduction domain was incorporated directly into the primary structure of the 53BP1. These complexes were efficiently taken up into human osteosarcoma cells and synchronously released from endocytic vesicles by brief exposure to far-red light in the presence of the photosensitizer, disulfonated aluminum phthalocyanine. Upon release, the streptavidin cargo entered the nucleus and was recruited to repair foci. 53BP1-based platforms provide a method for targeted, temporally controlled delivery of macromolecular agents to sites of double-strand break repair. With the delivery platforms, we are capable to visualize, modify and redirect DSB repair pathways by coupling various nanomaterials to study machinery or manipulate for therapy purpose in the future.

Modulating the tumor microenvironment to enhance efficacy of PARP inhibitors.

e14715 Background: PARP inhibitors (PARPi) have demonstrated activity in HR deficient tumors through synthetic lethality. However, their clinical efficacy has not been highly promising, with response rates ranging between 30-60% for Olaparib, Talazoparib and Veliparib (1). This highlights the necessity to understand the mechanisms of resistance and how these agents work in the context of the tumor microenvironment. In this study, we explore the effect of hypoxia on the efficacy of PARPi in a range of HR-deficient tumors. Hypoxia is known to downregulate genes of homologous recombination (2) but its role in HR deficiency in still unknown. Methods: HR deficient tumor cell lines were treated with PARPi under both aerobic and hypoxic conditions. DNA damage markers such as γH2AX, 53BP1 and RIF1 were detected by immunofluorescence. Tumor xenografts were grown to 100 mm3 prior to treatment with vehicle, PARPi, hypoxia activated cytotoxin, or the combination and tumor volumes were measured. Co-localization of apoptosis and hypoxia was detected by IHC. Finally, hypoxic signature in breast PDX models was correlated with their sensitivity to PARPi. Results: We found that hypoxic cells are significantly more resistant to PARPi in cell culture models. Efficiency of inhibition of PARP activity by these agents was similar under both air and hypoxia. The DNA damage marker γH2AX increased in cells treated in air, but not under hypoxia. There was no loss of 53BP1 protein that regulate engagement of the c-NHEJ pathway, indicating that HR has not been restored in these cells (3). However, it’s binding partner RIF1 was lost in hypoxia upon treatment indicating deficient signaling downstream of 53BP1. RIF1 is known to inhibit 5’ resection and its loss leads to resistance to PARPi in BRCA-deficient cells (4). In vivo, combining PARPi with hypoxia activated prodrugs led to substantial tumor growth delay in three xenograft models. DNA damage and apoptosis in these tumors was only detected outside of hypoxic regions positive for pimonidazole. In the breast PDX models, there was a strong correlation between the level of hypoxia in these tumors and their sensitivity to Olaparib. Conclusions: The significant increase in anti-tumor effects with the combination of PARPi and hypoxia-targeting cytotoxins leads us to propose a new strategy for tumors which are not responsive to PARPi alone. 1. Brown JS et al. Br J cancer 2016, 114, 713-5 2. Chan N et al. Cancer Res 2010, 70,8045-54 3. Bunting SF et al. Cell 2010, 141, 243-54 4. Chapman JR et al. Mol Cell 2013, 49, 858-71.

A role of the 53BP1 protein in genome protection: structural and functional characteristics of 53BP1-dependent DNA repair.

Nuclear architecture plays a significant role in DNA repair mechanisms. It is evident that proteins involved in DNA repair are compartmentalized in not only spontaneously occurring DNA lesions or ionizing radiation-induced foci (IRIF), but a specific clustering of these proteins can also be observed within the whole cell nucleus. For example, 53BP1-positive and BRCA1-positive DNA repair foci decorate chromocenters and can appear close to nuclear speckles. Both 53BP1 and BRCA1 are well-described factors that play an essential role in double-strand break (DSB) repair. These proteins are members of two protein complexes: 53BP1-RIF1-PTIP and BRCA1-CtIP, which make a “decision” determining whether canonical nonhomologous end joining (NHEJ) or homology-directed repair (HDR) is activated. It is generally accepted that 53BP1 mediates the NHEJ mechanism, while HDR is activated via a BRCA1-dependent signaling pathway. Interestingly, the 53BP1 protein appears relatively quickly at DSB sites, while BRCA1 is functional at later stages of DNA repair, as soon as the Mre11-Rad50-Nbs1 complex is recruited to the DNA lesions. A function of the 53BP1 protein is also linked to a specific histone signature, including phosphorylation of histone H2AX (γH2AX) or methylation of histone H4 at the lysine 20 position (H4K20me); therefore, we also discuss an epigenetic landscape of 53BP1-positive DNA lesions.

LC8/DYNLL1 is a 53BP1 effector and regulates checkpoint activation.

The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.

Lipolytic inhibitor G0S2 modulates glioma stem-like cell radiation response

BackgroundIonizing radiation (IR) therapy is the standard first-line treatment for newly diagnosed patients with glioblastoma (GBM), the most common and malignant primary brain tumor. However, the effects of IR are limited due to the aberrant radioresistance of GBM.MethodsTranscriptome analysis was performed using RNA-seq in radioresistant patient-derived glioma stem-like cells (GSCs). Survival of glioma patient and mice bearing-brain tumors was analyzed by Kaplan–Meier survival analysis. Lipid droplet and γ-H2AX foci-positive cells were evaluated using immunofluorescence staining.ResultsLipolytic inhibitor G0/G1 switch gene 2 (G0S2) is upregulated in radioresistant GSCs and elevated in clinical GBM. GBM patients with high G0S2 expression had significantly shorter overall survival compared with those with low expression of G0S2. Using genetic approaches targeting G0S2 in glioma cells and GSCs, we found that knockdown of G0S2 promoted lipid droplet turnover, inhibited GSC radioresistance, and extended survival of xenograft tumor mice with or without IR. In contrast, overexpression of G0S2 promoted glioma cell radiation resistance. Mechanistically, high expression of G0S2 reduced lipid droplet turnover and thereby attenuated E3 ligase RNF168-mediated 53BP1 ubiquitination through activated the mechanistic target of rapamycin (mTOR)-ribosomal S6 kinase (S6K) signaling and increased 53BP1 protein stability in response to IR, leading to enhanced DNA repair and glioma radioresistance.ConclusionsOur findings uncover a new function for lipolytic inhibitor G0S2 as an important regulator for GSC radioresistance, suggesting G0S2 as a potential therapeutic target for treating gliomas.

Shieldin - the protector of DNA ends.

DNA double-strand breaks are a threat to genome integrity and cell viability. The nucleolytic processing of broken DNA ends plays a central role in dictating the repair processes that will mend these lesions. Usually, DNA end resection promotes repair by homologous recombination, whereas minimally processed ends are repaired by non-homologous end joining. Important in this process is the chromatin-binding protein 53BP1, which inhibits DNA end resection. How 53BP1 shields DNA ends from nucleases has been an enduring mystery. The recent discovery of shieldin, a four-subunit protein complex with single-stranded DNA-binding activity, illuminated a strong candidate for the ultimate effector of 53BP1-dependent end protection. Shieldin consists of REV7, a known 53BP1-pathway component, and three hitherto uncharacterized proteins: C20orf196 (SHLD1), FAM35A (SHLD2), and CTC-534A2.2 (SHLD3). Shieldin promotes many 53BP1-associated activities, such as the protection of DNA ends, non-homologous end joining, and immunoglobulin class switching. This review summarizes the identification of shieldin and the various models of shieldin action and highlights some outstanding questions requiring answers to gain a full molecular understanding of shieldin function.

Molecular biomarkers of DNA damage in diffuse large-cell lymphoma—a review

Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive form of non-Hodgkin’s lymphoma with a long-term survival rate of around 60% upon standard treatment with rituximab and chemotherapy. While this marks a great improvement over the pre-rituximab/pre-biologicals era, DLBCLs remain a heterogeneous disease with marked variability in clinical outcome. Efforts have been undertaken to predict its course based on characteristics such as the activated B-cell (ABC) versus germinal centre B-cell (GCB) types, but so far none of these has had much impact on treatment and outcome, and the international prognostic index, based on clinical and basic laboratory features, remains the best risk predictor. This review focuses on 13 biomarkers that have been described in the context of diagnostic tests, clinical studies or basic research related to solid and haematological malignancies in general or DLBCL in particular. These include biomarkers of the DNA repair response such as the phosphorylated histone variant 2AX (γH2AX), p53 binding protein (53BP1), Ataxia telangiectasia mutated protein (ATM), Ataxia telangiectasia and Rad3-related protein (ATR), the DNA-dependent protein kinase (DNA-PK) and Rad51 as well as proto-oncogene products like Myc, B-cell lymphoma 2 (Bcl-2), breast cancer 1 (BRCA1) and signal transducer and activator of transcription 3 (STAT3). We have also included 8-Hydroxy-2'-desoxyguanosine (8-OHdG), a marker for oxidative stress, and discuss a link between two proteins involved in apoptosis, the proteasome activator 28γ (PA28γ) and tumour suppressor p53. Our investigation revealed a connection between almost all biomarkers, which are based on methods such as indirect immunofluorescence, fluorescence in-situ hybridization or immunohistochemistry.