Levels Of 53BP1 Research Articles

Radiosensitizing effects of CDK4/6 inhibitors in hormone receptor-positive and HER2-negative breast cancer mediated downregulation of DNA repair mechanism and NF-κB-signaling pathway

CDK4/6 inhibitors combined with endocrine therapy prolonged survival in hormone receptor (HR)-positive and HER2-negative advanced breast cancer. We investigated whether CDK4/6 inhibitors enhance radiosensitivity and their underlying mechanisms of this subtype of breast cancer. In vitro and in vivo experiments were conducted using two HR-positive and HER2-negative breast cancer cell lines (MCF-7 and T-47D), CDK4/6 inhibitors (ribociclib and palbociclib) and radiotherapy (RT) to assess the biological functions and mechanisms. The radiation-enhancing effect was assessed using clonogenic assays; γH2AX and 53BP1 levels were assessed by immunofluorescence to evaluate DNA damage. The levels of phospho (p)-ERK, c-Myc, and DNA-double strand break (DSB)-related molecules, p-DNA-PKcs, Rad51, and p-ATM, were assessed by western blotting. We used an NF-κB p65 transcription factor assay kit to evaluate NF-κB activity. We evaluated the antitumor effect of the combination of RT and ribociclib through the MCF-7 orthotopic xenograft model. The synergistic effects of combining RT with ribociclib and palbociclib pretreatment were demonstrated by clonogenic assay. CDK4/6 inhibitors synergistically increased the numbers of RT-induced γH2AX and 53BP1, downregulated the expression of p-DNA-PKcs, Rad51 and p-ATM activated by RT, and reduced RT-triggering p-ERK expression, NF-κB activation, and its down-streaming gene, c-Myc. Combined ribociclib and RT reduced the growth of MCF-7 cell xenograft tumors, and downregulated the immunohistochemical expression of p-ERK, p-NF-κB p65, and c-Myc compared to that in the control group. Combining CDK4/6 inhibitors enhanced radiosensitivity of HR-positive and HER2-negative breast cancer cells at least by reducing DNA-DSB repair and weakening the activation of ERK and NF-κB signaling by RT.

53BP1 loss elicits cGAS-STING-dependent antitumor immunity in ovarian and pancreatic cancer

53BP1 nucleates the anti-end resection machinery at DNA double-strand breaks, thereby countering BRCA1 activity. Loss of 53BP1 leads to DNA end processing and homologous recombination in BRCA1-deficient cells. Consequently, BRCA1-mutant tumors, typically sensitive to PARP inhibitors (PARPi), become resistant in the absence of 53BP1. Here, we demonstrate that the ‘leaky’ DNA end resection in the absence of 53BP1 results in increased micronuclei and cytoplasmic double-stranded DNA, leading to activation of the cGAS-STING pathway and pro-inflammatory signaling. This enhances CD8+ T cell infiltration, activates macrophages and natural killer cells, and impedes tumor growth. Loss of 53BP1 correlates with a response to immune checkpoint blockade (ICB) and improved overall survival. Immunohistochemical assessment of 53BP1 in two malignancies, high grade serous ovarian cancer and pancreatic ductal adenocarcinoma, which are refractory to ICBs, reveals that lower 53BP1 levels correlate with an increased adaptive and innate immune response. Finally, BRCA1-deficient tumors that develop resistance to PARPi due to the loss of 53BP1 are susceptible to ICB. Therefore, we conclude that 53BP1 is critical for tumor immunogenicity and underpins the response to ICB. Our results support including 53BP1 expression as an exploratory biomarker in ICB trials for malignancies typically refractory to immunotherapy.

Radiosensitization effect of quinoline-indole-schiff base derivative 10E on non-small cell lung cancer cells in vitro and in tumor xenografts.

Radioresistance is an inevitable obstacle in the clinical treatment of inoperable patients with non-small cell lung cancer (NSCLC). Combining treatment with radiosensitizers may improve the efficacy of radiotherapy. Previously, the quinoline derivative 10E as new exporter of Nur77 has shown superior antitumor activity in hepatocellular carcinoma. Here, we aimed to investigate the radiosensitizing activity and acting mechanisms of 10E. In vitro, A549 and H460 cells were treated with control, ionizing radiation (IR), 10E, and 10E + IR. Cell viability, apoptosis, and cycle were examined using CCK-8 and flow cytometry assays. Protein expression and localization were examined using western blotting and immunofluorescence. Tumor xenograft models were established to evaluate the radiosensitizing effect of 10E in vivo. 10E significantly inhibited cell proliferation and increased their radiosensitivity while reducing level of p-BCRA1, p-DNA-PKs, and 53BP1 involved in the DNA damage repair pathway, indicating that its radiosensitizing activity is closely associated with repressing DNA damage repair. A549 cells showed low level of Nur77 and a low response to IR but 10E-treated A549 cells showed high level of Nur77 indicating that Nur77 is a core radiosensitivity factor and 10E restores the expression of Nur77. Nur77 and Ku80 extranuclear co-localization in the 10E-treated A549 cells suggested that 10E-modulated Nur77 nuclear exportation inhibits DNA damage repair pathways and increases IR-triggered apoptosis. The combination of 10E and IR significantly inhibits tumor growth in a tumor xenograft model. Our findings suggest that 10E acts as a radiosensitizer and that combining 10E with radiotherapy may be a potential strategy for NSCLC treatment.

53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication

RNA-binding proteins (RBPs) are found at replication forks, but their direct interaction with DNA-embedded RNA species remains unexplored. Here, we report that p53-binding protein 1 (53BP1), involved in the DNA damage and replication stress response, is an RBP that directly interacts with Okazaki fragments in the absence of external stress. The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments. Conversely, depletion of FEN1, resulting in the accumulation of uncleaved RNA primers, increases 53BP1 levels at replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S-phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1 is anchored at nascent DNA through its RNA-binding activity, highlighting the role of an RNA-protein interaction at replication forks.

Pilot clinical trial and phenotypic analysis in chemotherapy-pretreated, metastatic triple-negative breast cancer patients treated with oral TAK-228 and TAK-117 (PIKTOR) to increase DNA damage repair deficiency followed by cisplatin and nab paclitaxel

BackgroundA subset of triple-negative breast cancers (TNBCs) have homologous recombination deficiency with upregulation of compensatory DNA repair pathways. PIKTOR, a combination of TAK-228 (TORC1/2 inhibitor) and TAK-117 (PI3Kα inhibitor), is hypothesized to increase genomic instability and increase DNA damage repair (DDR) deficiency, leading to increased sensitivity to DNA-damaging chemotherapy and to immune checkpoint blockade inhibitors.Methods10 metastatic TNBC patients received 4 mg TAK-228 and 200 mg TAK-117 (PIKTOR) orally each day for 3 days followed by 4 days off, weekly, until disease progression (PD), followed by intravenous cisplatin 75 mg/m2 plus nab paclitaxel 220 mg/m2 every 3 weeks for up to 6 cycles. Patients received subsequent treatment with pembrolizumab and/or chemotherapy. Primary endpoints were objective response rate with cisplatin/nab paclitaxel and safety. Biopsies of a metastatic lesion were collected prior to and at PD on PIKTOR. Whole exome and RNA-sequencing and reverse phase protein arrays (RPPA) were used to phenotype tumors pre- and post-PIKTOR for alterations in DDR, proliferation, and immune response.ResultsWith cisplatin/nab paclitaxel (cis/nab pac) therapy post PIKTOR, 3 patients had clinical benefit (1 partial response (PR) and 2 stable disease (SD) ≥ 6 months) and continued to have durable benefit in progression-free survival with pembrolizumab post-cis/nab pac for 1.2, 2, and 3.6 years. Their post-PIKTOR metastatic tissue displayed decreased mismatch repair (MMR), increased tumor mutation burden, and significantly lower levels of 53BP1, DAG Lipase β, GCN2, AKT Ser473, and PKCzeta Thr410/403 compared to pre-PIKTOR tumor tissue.ConclusionsPriming patients’ chemotherapy-pretreated metastatic TNBC with PIKTOR led to very prolonged response/disease control with subsequent cis/nab pac, followed by pembrolizumab, in 3 of 10 treated patients. Our multi-omics approach revealed a higher number of genomic alterations, reductions in MMR, and alterations in immune and stress response pathways post-PIKTOR in patients who had durable responses.Trial RegistrationThis clinical trial was registered on June 21, 2017, at ClinicalTrials.gov using identifier NCT03193853.

DIPG-24. BRD4 INHIBITION AS A RADIOSENSITIZER THROUGH BLOCKING DNA REPAIR FOR THE TREATMENT OF DIFFUSE MIDLINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) is one of the devastating childhood cancers. Radiation therapy (RT) remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance RT efficacy by combining it with radiosensitizing agents, though none have been successful in doing so. Given this, there is a critical need to identify effective therapeutics to enhance the RT anti-tumor activity in DIPG. Here, we identified BRD4 inhibitors as candidate radiosensitizers from a high throughput drug screening. DIPG cells show increased H3 K27 acetylation (H3K27ac) levels, which bind to BET bromodomain protein 4 (BRD4) and are strongly associated with active transcription. We tested two BRD4 inhibitors (BRD4i: AZD5153 and JQ-1) as well as genetic BRD4 depletion, and observed enhancement of radiation-induced DNA damage, confirming BRD4i as potential radiosensitizers in the treatment of DIPG. We evaluated the effects of BRD4i on gene expression using RNAseq, and observed significant inhibition of DNA-repair proteins such as BRCA1 and RAD51. CUT-RUN qPCR showed decreased H3K27ac at BRCA1 and RAD51 promoters. Combination of BRD4i and RT inhibited cell viability significantly using clonogenic survival assay, apoptosis assay, and also showed cell cycle arrest. Radiation-induced DNA double-strand break (DSB) repair was prolonged with BRD4i with high levels of gH2AX and 53BP1 likely due to inhibition of DNA-repair. In vivo studies revealed increased survival of animals treated with combination therapy of RT and BRD4i in comparison to either monotherapy. Together, these results highlight BRD4i as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

Assessment of Individual Radiosensitivity in Breast Cancer Patients Using a Combination of Biomolecular Markers

About 5% of patients undergoing radiotherapy (RT) develop RT-related side effects. To assess individual radiosensitivity, we collected peripheral blood from breast cancer patients before, during and after the RT, and γH2AX/53BP1 foci, apoptosis, chromosomal aberrations (CAs) and micronuclei (MN) were analyzed and correlated with the healthy tissue side effects assessed by the RTOG/EORTC criteria. The results showed a significantly higher level of γH2AX/53BP1 foci before the RT in radiosensitive (RS) patients in comparison to normal responding patients (NOR). Analysis of apoptosis did not reveal any correlation with side effects. CA and MN assays displayed an increase in genomic instability during and after RT and a higher frequency of MN in the lymphocytes of RS patients. We also studied time kinetics of γH2AX/53BP1 foci and apoptosis after in vitro irradiation of lymphocytes. Higher levels of primary 53BP1 and co-localizing γH2AX/53BP1 foci were detected in cells from RS patients as compared to NOR patients, while no difference in the residual foci or apoptotic response was found. The data suggested impaired DNA damage response in cells from RS patients. We suggest γH2AX/53BP1 foci and MN as potential biomarkers of individual radiosensitivity, but they need to be evaluated with a larger cohort of patients for clinics.

Intracellular calprotectin (S100A8/A9) facilitates DNA damage responses and promotes apoptosis in head and neck squamous cell carcinoma

ObjectivesIn head and neck squamous cell carcinoma (HNSCC), poor prognosis and low survival rates are associated with downregulated calprotectin. Calprotectin (S100A8/A9) inhibits cancer cell migration and invasion and facilitates G2/M cell cycle arrest. We investigated whether S100A8/A9 regulates DNA damage responses (DDR) and apoptosis in HNSCC after chemoradiation. Materials and methodsHuman HNSCC cases in TCGA were analyzed for relationships between S100A8/A9 and expression of apoptosis-related genes. Next, S100A8/A9-expressing and non-expressing carcinoma lines (two different lineages) were exposed to genotoxic agents and assessed for 53BP1 and γH2AX expression and percent of viable/dead cells. Finally, S100A8/A9-wild-type and S100A8/A9null C57BL/6j mice were treated with 4-NQO to induce oral dysplastic and carcinomatous lesions, which were compared for levels of 53BP1. ResultsIn S100A8/A9-high HNSCC tumors, apoptosis-related caspase family member genes were upregulated, whereas genes limiting apoptosis were significantly downregulated based on TCGA analyses. After X-irradiation or camptothecin treatment, S100A8/A9-expressing carcinoma cells (i.e., TR146 and KB-S100A8/A9) showed significantly higher 53BP1 and γH2AX expression, DNA fragmentation, proportions of dead cells, and greater sensitivity to cisplatin than wild-type KB or TR146-S100A8/A9-KD cells. Interestingly, KB-S100A8/A9Δ113-114 cells showed similar 53BP1 and γH2AX levels to S100A8/A9-negative KB and KB-EGFP cells. After 4-NQO treatment, 53BP1 expression in oral lesions was significantly greater in calprotectin+/+ than S100A8/A9null mice. ConclusionsIn HNSCC cells, intracellular calprotectin is strongly suggested to potentiate DDR and promote apoptosis in response to genotoxic agents. Hence, patients with S100A8/A9-high HNSCC may encounter more favorable outcomes because more tumor cells enter apoptosis with increased sensitivity to chemoradiation therapy.

Incorporation of 53BP1 into phase-separated bodies in cancer cells during aberrant mitosis.

53BP1 (also known as TP53BP1) is a key mediator of the non-homologous end joining (NHEJ) DNA repair pathway, which is the primary repair pathway in interphase cells. However, the mitotic functions of 53BP1 are less well understood. Here, we describe 53BP1 mitotic stress bodies (MSBs) formed in cancer cell lines in response to delayed mitosis. These bodies displayed liquid-liquid phase separation characteristics, were close to centromeres, and included lamin A/C and the DNA repair protein RIF1. After release from mitotic arrest, 53BP1 MSBs decreased in number and moved away from the chromatin. Using GFP fusion constructs, we found that the 53BP1 oligomerization domain region was required for MSB formation, and that inclusion of the 53BP1 N terminus increased MSB size. Exogenous expression of 53BP1 did not increase MSB size or number but did increase levels of MSB-free 53BP1. This was associated with slower mitotic progression, elevated levels of DNA damage and increased apoptosis, which is consistent with MSBs suppressing a mitotic surveillance by 53BP1 through sequestration. The 53BP1 MSBs, which were also found spontaneously in a subset of normally dividing cancer cells but not in non-transformed cells (ARPE-19), might facilitate the survival of cancer cells following aberrant mitoses. This article has an associated First Person interview with the first author of the paper.

EXTH-29. RADIOSENSITIZATION BY BRD4 INHIBITION IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG), the most frequent brainstem tumor in pediatrics, is one of the devastating childhood cancers, and virtually all patients die within two years after diagnosis. Since these tumors occur in the brainstem which is vital area, there are no surgical options for providing relief to patients, and conventional chemotherapy. Radiation therapy (RT) remains the only effective treatment. Radiosensitization is a mean to improve the therapeutic balance between efficacy and toxicity of RT, but not used in clinically. Here, we found BRD4 inhibitor as a radiosensitizer from drug screening. DIPG shows increased H3 K27 acetylation (H3K27ac), which binds to BET bromodomain protein 4 (BRD4) and they are strongly associated with active transcription. We tested two BRD4 inhibiter (BRD4i : AZD5153 and JQ-1) and genetic BRD4 depletion enhances radiation-induced DNA damage, making it a potential radiosensitizer in the treatment of DIPG. We evaluated the effects of BRD4i on genes expression using RNA sequence, inhibited significantly DNA-repair proteins such as BRCA1 and RAD51. CUT-RUN qPCR showed decreased H3K27ac at BRCA1 and RAD51. Combination with BRD4i and RT inhibited cell viability significantly in clonogenic survival assay, apoptosis assay, and showed cell cycle arrest. Radiation-induced DNA double-strand break (DSB) repair was prolonged high level of rH2AHX and 53BP1 with BRD4i because of inhibited DNA-repair. In vivo studies revealed increased survival of animals treated with combination therapy of RT and BRD4i in compared to either monotherapy. Together, these results highlight BRD4i as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

Characterization of a Knock-In Mouse Model with a Huntingtin Exon 1 Deletion.

The Huntingtin (HTT) N-terminal domains encoded by Huntingtin's (HTT) exon 1 consist of an N17 domain, the polyglutamine (polyQ) stretch and a proline-rich region (PRR). These domains are conserved in mammals and have been hypothesized to modulate HTT's functions in the developing and adult CNS, including DNA damage repair and autophagy. This study longitudinally characterizes the in vivo consequences of deleting the murine Htt N-terminal domains encoded by Htt exon 1. Knock-in mice with a deletion of Htt exon 1 sequences (HttΔE1) were generated and bred into the C57BL/6J congenic genetic background. Their behavior, DNA damage response, basal autophagy, and glutamatergic synapse numbers were evaluated. Progeny from HttΔE1/+ intercrosses are born at the expected Mendelian frequency but with a distorted male to female ratio in both the HttΔE1/ΔE1 and Htt+/+ offspring. HttΔE1/ΔE1 adults exhibit a modest deficit in accelerating rotarod performance, and an earlier increase in cortical and striatal DNA damage with elevated neuronal pan-nuclear 53bp1 levels compared to Htt+/+ mice. However, a normal response to induced DNA damage, normal levels of basal autophagy markers, and no significant differences in corticocortical, corticostriatal, thalamocortical, or thalamostriatal synapses numbers were observed compared to controls. Our results suggest that deletion of the Htt N-terminus encoded by the Htt exon 1 does not affect Htt's critical role during embryogenesis, but instead, may have a modest effect on certain motor tasks, basal levels of DNA damage in the brain, and Htt function in the testis.

Apoptosis and DNA Repair in Irradiated Cerebral Organoids

In the treatment of CNS neoplasms, radiation therapy to high biologically effective doses is performed in both fractionated and single fraction schemes. Inevitably, normal CNS tissues are irradiated, leading to both acute and late toxicities, and demanding greater insight into normal tissue effects and development of innovative strategies for radioprotection and mitigation. For this purpose, the cerebral organoid, which recapitulates many cell types and complex architectures of the human brain, is proposed as a model system. The objective of our study was to characterize the response of cerebral organoids to high single fraction doses of photon irradiation in terms of apoptosis, DNA damage, and activation of DNA repair.Cerebral organoids were prepared from human embryonic stem cells as previously described. CNS tissue differentiation was verified by histopathology. Irradiation of organoids was accomplished using a 160 kV x-ray irradiator. Apoptosis was characterized by immunofluorescence of cleaved caspase 3 and TUNEL assay. DNA damage and repair was determined by immunofluorescence of γH2Ax foci and 53BP1.While cerebral organoids demonstrated no gross morphologic changes to high single dose radiation up to 40 Gy even when incubated for several months following treatment, individual apoptotic cells were readily apparent at 72 hours after 20 Gy. High levels of γH2Ax foci and 53BP1 were observed within hours. While characterization of the kinetics of DNA repair showed significant resolution of DNA damage foci by 24 hours, many foci persisted at 72 hours alongside activation of apoptotic pathways. Choroid plexus tissue was highly resistant to radiation-induced apoptosis.Cerebral organoids provide a viable experimental platform to study effects of radiation in normal CNS tissues, and may potentially aid in the development of neuroprotective strategies.

P85β alters response to EGFR inhibitor in ovarian cancer through p38 MAPK-mediated regulation of DNA repair

EGFR signaling promotes ovarian cancer tumorigenesis, and high EGFR expression correlates with poor prognosis. However, EGFR inhibitors alone have demonstrated limited clinical benefit for ovarian cancer patients, owing partly to tumor resistance and the lack of predictive biomarkers. Cotargeting EGFR and the PI3K pathway has been previously shown to yield synergistic antitumor effects in ovarian cancer. Therefore, we reasoned that PI3K may affect cellular response to EGFR inhibition. In this study, we revealed PI3K isoform-specific effects on the sensitivity of ovarian cancer cells to the EGFR inhibitor erlotinib. Gene silencing of PIK3CA (p110α) and PIK3CB (p110β) rendered cells more susceptible to erlotinib. In contrast, low expression of PIK3R2 (p85β) was associated with erlotinib resistance. Depletion of PIK3R2, but not PIK3CA or PIK3CB, led to increased DNA damage and reduced level of the nonhomologous end joining DNA repair protein BRD4. Intriguingly, these defects in DNA repair were reversed upon erlotinib treatment, which caused activation and nuclear import of p38 MAPK to promote DNA repair with increased protein levels of 53BP1 and BRD4 and foci formation of 53BP1. Remarkably, inhibition of p38 MAPK or BRD4 re-sensitized PIK3R2-depleted cells to erlotinib. Collectively, these data suggest that p38 MAPK activation and the subsequent DNA repair serve as a resistance mechanism to EGFR inhibitor. Combined inhibition of EGFR and p38 MAPK or DNA repair may maximize the therapeutic potential of EGFR inhibitor in ovarian cancer.

Inhibition of tryptophan 2,3-dioxygenase impairs DNA damage tolerance and repair in glioma cells.

Expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy in gliomas through kynurenine (KYN) signaling. We report that inhibition of TDO activity attenuated recovery from replication stress and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU). Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to BCNU treatment, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced—indicative of increased fork collapse. Analysis of quantitative proteomic results revealed that TDO inhibition reduced nuclear 53BP1 and sirtuin levels. We confirmed that cells lacking TDO activity exhibited elevated gamma-H2AX signal and defective recruitment of 53BP1 to chromatin following BCNU treatment, which corresponded with delayed repair of DNA breaks. Addition of exogenous KYN increased the rate of break repair. TDO inhibition diminished SIRT7 deacetylase recruitment to chromatin, which increased histone H3K18 acetylation—a key mark involved in preventing 53BP1 recruitment to sites of DNA damage. TDO inhibition also sensitized cells to ionizing radiation (IR)-induced damage, but this effect did not involve altered 53BP1 recruitment. These experiments support a model where TDO-mediated KYN signaling helps fuel a robust response to replication stress and DNA damage.

DNA damage response and apoptosis induced by hyperthermia in human umbilical cord blood lymphocytes

While hyperthermia (HT) is a promising modality for cancer treatment, the knowledge on mechanisms of its effect on cells is still limited. We have investigated DNA double-strand break (DSB) and apoptosis induced by HT. Umbilical cord blood lymphocytes (UCBL) were subjected to HT at 43 °C. We have treated cells for 1 h (1 h HT), 2 h (2 h HT) and by combined HT and ice treatment (both lasting 1 h). Enumeration of DSB by 53BP1/γH2AX DNA repair focus formation and early apoptosis by γH2AX pan-staining was conducted by automated fluorescent microscopy. Apoptotic stages and viability were assessed by the annexin/propidium iodide (PI) assay using flow cytometry 0, 18, and 42 h post-treatment. HT induced either immediate (2 h HT) or postponed (1 h HT) DNA damage. The levels of 53BP1 and γH2AX foci differed under the same treatment conditions, suggesting that the ratio of co-localized γH2AX/53BP1 foci to all γH2AX and also to all 53BP1 foci could be a valuable marker. The ratio of co-localized foci increased immediately after 2 h HT regardless the way of assessment. For the first time we show, by both annexin/PI and γH2AX pan-staining assay that apoptosis can be induced during or immediately after the 2 h HT treatment. Our results suggest that HT may induce DSB in dependence on treatment duration and post-treatment time due to inhibition of DNA repair pathways and that HT-induced apoptosis might be dependent or associated with DSB formation in human lymphocytes. Assessment of γH2AX pan-staining in lymphocytes affected by HT may represent a valuable marker of HT treatment side effects.

Involvement of a novel regulatory cascade consisting of SET-H3K18ac/H3K27ac-53BP1 in Cr(VI)-induced malignant transformation of 16HBE cells

Hexavalent chromium (Cr(VI)) is a well-established human carcinogen with DNA damaging effects. Recently we established a Cr(VI)-induced malignant transformation model from a human bronchial epithelial (16HBE) cell line, and in the transformed (16HBE-T) cells reduced levels of 53BP1 (critical for DNA repair) and the acetylated histone H3K18/27 (H3K18/27ac) were observed. In 16HBE-T cells SET (a multifunctional protein) was elevated by Cr(VI) through quantitative proteomics analysis. In the present study, we further explore the involvement of SET in the H3K18/27ac/53BP1 cascade in the 16HBE-T model, primarily by knockdown of SET. Bioinformatic analysis of the differentially expressed proteins indicated enrichment in histone modifications, in which SET was a major regulator. In 16HBE cells SET expression was enhanced by Cr(VI) in a concentration- and exposure duration-dependent manner. In 16HBE-T cells, SET knockdown showed the following effects: reversal of H3K18/27ac and 53BP1 levels, enhanced enrichment H3K18/27ac in 53BP1′s promotor region, increase rate of apoptosis and cell cycle G0/G1 arrest (with or without Cr(VI) treatment), and reduced colony-forming efficiency. Finally, In comparison with benzo(a)pyrene-transformed (malignant, 16HBE-B) cells from 16HBE where no changes in H3K18/27ac, 53BP1 or SET were observed, while the H3K18/27ac/53BP1 cascade was downregulated and SET upregulated in 16HBE-T cells, as compared with the parental 16HBE cells; thus the changes in 16HBE-T might be a specific effect of Cr(VI). In conclusion, our results suggest that SET may be involved in the malignant cell transformation, through inhibiting the H3K18/27ac/53BP1 cascade, at least in the 16HBE cell model.

Considering Cell Proliferation to Optimize Detection of Radiation-Induced 53BP1 Positive Foci in 15 Mouse Strains Ex Vivo.

Due to high metabolic activity, proliferating cells continuously generate free radicals, which induce DNA double-strand breaks (DSB). Fluorescently tagged nuclear foci of DNA repair protein 53 binding protein-1 (53BP1) are used as a standard metric for measuring DSB formation at baseline and in response to environmental insults such as radiation. Here we demonstrate that the background level of spontaneous 53BP1+ foci formation can be modeled mathematically as a function of cell confluence, which is a metric of their proliferation rate. This model was validated using spontaneous 53BP1+ foci data from 72 cultures of primary skin fibroblasts derived from 15 different strains of mice, showing a ∼10-fold decrease from low to full confluence that is independent of mouse strain. On the other hand, the baseline level of spontaneous 53BP1+ foci in a fully confluent cell population was strain-dependent, suggesting genomic associations, and correlated with radiation sensitivity based on previous measurements in the same cell lines. Finally, we have developed an online open-access tool to correct for the effect of cell confluence on 53BP1+ foci-based quantification of DSB. This tool provides guidelines for the number of cells required to reach statistical significance for the detection of DSB induced by low doses of ionizing radiation as a function of confluence and time postirradiation.

BRCA1 Expression and Outcome in Patients With EGFR-Mutant NSCLC Treated With Gefitinib Alone or in Combination With Olaparib

IntroductionDNA repair capacity, as exemplified by BRCA1 gene expression, is related with outcome to EGFR tyrosine kinase inhibitors in patients with EGFR-mutant NSCLC. Olaparib, a PARP inhibitor, reduces BRCA1 expression. Olaparib was tested in combination with gefitinib versus gefitinib single agent, as a first-line therapy for patients with EGFR-mutant NSCLC in the GOAL study (trial registration: NCT01513174). Here, we report the results of the biomarker-related prespecified secondary objectives of the GOAL study. MethodsWe evaluated the impact of BRCA1 mRNA expression in 91 patients with EGFR-mutant NSCLC. Of those 91 patients, 51 were randomized to treatment with gefitinib and 40 were randomized to treatment with gefitinib plus olaparib. We explored in vitro whether BRCA1 mRNA levels are related with outcome to gefitinib plus olaparib. The expression levels of 53BP1, CtIP, and AXL were also explored and correlated with the treatment outcome. ResultsOverall, as what happened in the GOAL study, no statistically significant difference was observed in median progression-free survival (PFS) between the two treatment arms, for the 91 patients of the present study (p = 0.2419). For patients with high BRCA1 mRNA expression (BRCA1-high group), median PFS was 12.9 months in the gefitinib plus olaparib arm, compared with 9.2 months in the gefitinib arm (p = 0.0449). In the gefitinib arm, median PFS was 9.1 months for the BRCA1-high group and 10.2 months for the BRCA1-low group (p = 0.0193). We observed a more pronounced synergism of gefitinib plus olaparib in cells with higher BRCA1 compared with those with low BRCA1 mRNA expression. ConclusionsHigh BRCA1 mRNA expression identified patients with NSCLC who benefited from gefitinib plus olaparib in the GOAL phase 2 clinical trial.

DNA damage response and preleukemic fusion genes induced by ionizing radiation in umbilical cord blood hematopoietic stem cells

There is clear evidence that ionizing radiation (IR) causes leukemia. For many types of leukemia, the preleukemic fusion genes (PFG), as consequences of DNA damage and chromosomal translocations, occur in hematopoietic stem and progenitor cells (HSPC) in utero and could be detected in umbilical cord blood (UCB) of newborns. However, relatively limited information is available about radiation-induced apoptosis, DNA damage and PFG formation in human HSPC. In this study we revealed that CD34+ HSPC compared to lymphocytes: (i) are extremely radio-resistant showing delayed time kinetics of apoptosis, (ii) accumulate lower level of endogenous DNA damage/early apoptotic γH2AX pan-stained cells, (iii) have higher level of radiation-induced 53BP1 and γH2AX/53BP1 co-localized DNA double stranded breaks, and (iv) after low dose of IR may form very low level of BCR-ABL PFG. Within CD34+ HSPC we identified CD34+CD38+ progenitor cells as a highly apoptosis-resistant population, while CD34+CD38− hematopoietic stem/multipotent progenitor cells (HSC/MPP) as a population very sensitive to radiation-induced apoptosis. Our study provides critical insights into how human HSPC respond to IR in the context of DNA damage, apoptosis and PFG.

53BP1 Accumulation in Circulating Tumor Cells Identifies Chemotherapy-Responsive Metastatic Breast Cancer Patients.

Evidence suggests that the DNA end-binding protein p53-binding protein 1 (53BP1) is down-regulated in subsets of breast cancer. Circulating tumor cells (CTCs) provide accessible “biopsy material” to track cell traits and functions and their alterations during treatment. Here, we prospectively monitored the 53BP1 status in CTCs from 67 metastatic breast cancer (MBC) patients with HER2- CTCs and known hormone receptor (HR) status of the primary tumor and/or metastases before, during, and at the end of chemotherapeutic treatment with Eribulin. Nuclear 53BP1 staining and genomic integrity were evaluated by immunocytochemical and whole-genome-amplification-based polymerase chain reaction (PCR) analysis, respectively. Comparative analysis of CTCs from patients with triple-negative and HR+ tumors revealed elevated 53BP1 levels in CTCs from patients with HR+ metastases, particularly following chemotherapeutic treatment. Differences in nuclear 53BP1 signals did not correlate with genomic integrity in CTCs at baseline or with nuclear γH2AX signals in MBC cell lines, indicating that 53BP1 detected features beyond DNA damage. Kaplan–Meier analysis revealed an increasing association between nuclear 53BP1-positivity and progression-free survival (PFS) during chemotherapy until the final visit. Our data suggest that 53BP1 detection in CTCs could be a useful marker to capture dynamic changes of chemotherapeutic responsiveness in triple-negative and HR+ MBC.