DNA DSB Repair Research Articles

Selected Articles from This Issue

In this First Disclosure, Murray and colleagues describe TPX-0131, a compact macrocyclic ALK inhibitor designed to fit completely in the ATP-binding pocket and inhibit both wild-typ. ALK and drug-resistance mutations. Using a combination of biochemical, cellular, and in vivo preclinical assessments, TPX-0131 was shown to a CNS-penetrant molecule that potently inhibits wild-type ALK and a broad array of clinically relevant ALK mutants that limit the utility of previous generations of ALK inhibitors such as solvent front, gatekeeper, hinge region, and compound mutations. TPX-0131 currently is being evaluated in a phase 1/2 clinical study of TKI-pretreated patients with ALK+ NSCLC (NCT04849273).This First Disclosure describes the lead selection and preclinical research supporting the development of SENTI-101, a novel allogeneic cell therapy preclinical product candidate for the treatment of peritoneal carcinomatosis, including advanced ovarian cancer. SENTI-101 uses mesenchymal stromal cells innate capacity to traffic and localize to solid tumors to deliver two potent immune stimulatory cytokines (IL-12 and IL-21) to the tumor microenvironment. Studies conducted by Senti Bio demonstrate that, with this approach, scientists safely turned “cold” tumors “hot” resulting in long term anti-tumor immunity and rendered them susceptible to checkpoint inhibition. SENTI-101 demonstrates anti-tumor activity and proposes a cell modality that may address an unmet need for patients with ovarian cancer.Tumor hypoxia is a barrier to radiotherapy. It has been reported that DNAp. inhibition preferentially sensitizes tumor cells to radiation under hypoxia in vitro. However, whether DNAPK inhibition sensitizes hypoxic tumor cells to radiation in vivo has not been reported. In this study, Jiang et al show that transient DNAPK inhibition by a novel selective DNAPK inhibitor, NU5455 favorably compromises the repair of radiation-induced DNA DSBs in chronically hypoxic tumor cells in vivo, suggesting that DNAPK inhibition may preferentially sensitizes chronically hypoxic tumor cells to radiotherapy, which could form the basis for clinical trials investigating the impact of DNAPK inhibitors in radioresistant hypoxic tumors.Therapy-resistant leukemic stem cells can lead to the relapse of acute myeloid leukemia. In this study, Lee and colleagues target these residual cancer cells using mucin domain 3 (TIM-3) targeted chimeric antigen receptor (CAR) T cells. TIM-3 was selected due to its high expression on AML blasts and leukemic stem cells and its low expression on normal hematopoietic cells. Phage display was used to determine a TIM-3 antibody suitable for expression on the CAR T cells, which were subsequently tested on murine models and primary AML blasts. Although the consequences of TIM-3 targeting require further investigation, the authors demonstrate favorable anti-tumor activity by TIM-3 CAR T cells.

ATR Inhibitor M6620 (VX-970) Enhances the Effect of Radiation in Non-Small Cell Lung Cancer Brain Metastasis Patient-Derived Xenografts.

M6620, a selective ATP-competitive inhibitor of the ATM and RAD3-related (ATR) kinase, is currently under investigation with radiation in patients with non-small cell lung cancer (NSCLC) brain metastases. We evaluated the DNA damage response (DDR) pathway profile of NSCLC and assessed the radiosensitizing effects of M6620 in a preclinical NSCLC brain metastasis model. Mutation analysis and transcriptome profiling of DDR genes and pathways was performed on NSCLC patient samples. NSCLC cell lines were assessed with proliferation, clonogenic survival, apoptosis, cell cycle, and DNA damage signaling and repair assays. NSCLC brain metastasis patient-derived xenograft models were used to assess intracranial response and overall survival. In vivo IHC was performed to confirm in vitro results. A significant portion of NSCLC patient tumors demonstrated enrichment of DDR pathways. DDR pathways correlated with lung squamous cell histology; and mutations in ATR, ATM, BRCA1, BRCA2, CHEK1, and CHEK2 correlated with enrichment of DDR pathways in lung adenocarcinomas. M6620 reduced colony formation after radiotherapy and resulted in inhibition of DNA DSB repair, abrogation of the radiation-induced G2 cell checkpoint, and formation of dysfunctional micronuclei, leading to enhanced radiation-induced mitotic death. The combination of M6620 and radiation resulted in improved overall survival in mice compared with radiation alone. In vivo IHC revealed inhibition of pChk1 in the radiation plus M6620 group. M6620 enhances the effect of radiation in our preclinical NSCLC brain metastasis models, supporting the ongoing clinical trial (NCT02589522) evaluating M6620 in combination with whole brain irradiation in patients with NSCLC brain metastases.

Abstract 2536: Mutational landscape of the bronchial epithelium of individuals at high risk for lung cancer

Abstract Insights into mutational burden of the airway epithelium, the field of cancerization, may play important role in stratifying individuals at different levels of risk for lung cancer. We hypothesized that cytologically normal bronchial epithelium of patients with lung cancer and those at high risk for lung cancer harbor driver mutations. We performed next generation sequence-based targeted CAPP-Seq (CAncer Personalized Profiling by deep Sequencing) and whole exome sequencing (WES) of DNA from the bronchial epithelial cells from the bronchial brushings of individuals at-risk for lung cancer and patients with lung cancer as well as matched lung tumor tissues. We used the Tammemagi risk model to calculate the risk for lung cancer. Both CAPPSeq and WES data analyses showed higher mutation burden in lung tumor tissues compared to bronchial brushings. Using targeted CAPPSeq, a total of 129 nonsynonymous mutations were identified in bronchial brushings. Among individuals without lung cancer, 9 oncogenic driver mutations were identified in 5 out of 13 high risk and 2 out of 7 low risk individuals. Among individuals with cancer, 13 out of 22 brushings harbored 11 driver mutations, of which mutations in TP53 and PTEN were identified in both tumor and brushing matched samples. Mutations detected in the brushings of individuals with and without cancer were TP53, PIK3CA, NOTCH1, KRAS, MET, CUL3, and NF1. Using WES on the DNA from the same brushings (low risk, n=9; high risk, n=8 and lung cancer, n=17), we identified 19478 mutations total, and 90 of that were also identified by CAPPSeq. WES identified 80% of the mutations that were identified by CAPPSeq with >5% variant allelic frequencies (VAF). Mutations identified by CAPPSeq had VAF as low as 0.2% and vast majority of the mutations identified by WES had VAF >5%. Out of 9 mutations in TP53 identified in the brushings by CAPPSeq, 4 were identified by WES. Mutation in KMT2D, a histone methylation enzyme and believed to be a tumor suppressor, was identified by WES in the brushings and the tumor tissues of patients with lung cancer as well as in one high risk brushings. Mutational signature profiling identified tobacco mutational signature in the bronchial epithelium and tobacco, APOBEC and DNA-DSB repair signatures in the tumor tissues. Copy number variation (CNV) data analysis revealed significantly higher CNV levels in tumor samples than brushing samples. Genome-wide CNA profiling identified regions significantly influenced by CNV, such as deletions in chromosome region 19p13.3, which was previously reported to be related to lung cancer. We identified mutations in cancer driver genes in the bronchial brushings of individuals with and without lung cancer. This field of cancerization effect may provide a tool for risk assessment for lung cancer. This research was funded by U01CA196405 to PPM. Citation Format: SM Jamshedur Rahman, Shilin Zhao, Shih-Kai Chu, Yong Zou, Angela B. Hui, Thomas P. Stricker, Chen Heidi, Maximilian Diehn, Pierre P. Massion. Mutational landscape of the bronchial epithelium of individuals at high risk for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2536.

Abstract IA-010: Deciphering DNA end resection at DSBs

Abstract Double stranded DNA break (DSB)s are repaired by two major mechanistically distinct pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). A decisive factor in the choice between DSB repair pathways is in the competition between DNA end protection (necessary for NHEJ) and DNA end resection (necessary for HR). As the ‘master regulator’ of DSB repair pathway choice, 53BP1, a chromatin-associated reader of epigenetic marks at DSBs, helps channel DSBs into the NHEJ pathway by restricting BRCA1-dependent end resection. 53BP1 nucleates the assembly of a higher order ensemble that includes the Shieldin complex, CST complex, and DNA polymerase (Pol) a-primase complex. Loss of BRCA1 provides a therapeutic opportunity as these tumors are exquisitely sensitive to inhibitors of poly (ADP-ribose) polymerase (PARP), and are also susceptible to platinum-based drugs. Loss of 53BP1 or any of the 53BP1-interacting proteins restores end resection and causes PARPi resistance in BRCA1-mutant tumors. We recently identified Dynein light chain like protein 1 (DYNLL1) as as anti-resection factor. Loss of DYNLL1 allowed DNA end resection and restored HR, thereby inducing resistance to platinum drugs and PARPi in BRCA1-mutant tumors. At the molecular level, DYNLL1 limited the nucleolytic resection of DNA ends by interacting with the resection machinery. Importantly, purified DYNLL1 interacted with MRE11 and inhibited its exonuclease activity in vitro. Now we observe that 53BP1 loss prevents DYNLL1 recruitment to DSBs and more broadly to chromatin. We hypothesize that 53BP1 inhibits MRE11-mediated DNA end resection by recruiting DYNLL1 and that DYNLL1 functions in parallel of Shieldin-CST to regulate DNA end resection and DSB repair pathway choice. We are investigating the crosstalk between the Shieldin-CST and DYNLL1-MRE11 arms of the 53BP1-dependent anti-DNA end resection machinery. Citation Format: Dipanjan Chowdhury. Deciphering DNA end resection at DSBs [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-010.

A Role for RAGE in DNA Double Strand Breaks (DSBs) Detected in Pathological Placentas and Trophoblast Cells.

Impaired DNA damage responses are associated with several diseases, including pregnancy complications. Recent research identified an ATM-kinase dependent function for the nuclear isoform of the receptor for advanced glycation end-products (RAGE) during double strand break (DSB)-repair. RAGE contributes to end-resectioning of broken DNA sites by binding with the MRE11-Rad50-Nbs1 (MRN) complex. Placental research is limited regarding the impact of genomic instability and the mechanism for potential repair. We tested the hypothesis regarding the involvement of RAGE during the repair of placental DNA-DSBs. We first identified that the pregnancy complications of PE and preterm labor (PTL) experience loss of genomic integrity and an in vitro trophoblast cell model was used to characterize trophoblast DSBs. Colocalized immunofluorescence of γ-H2AX and RAGE support the potential involvement of RAGE in cellular responses to DNA-DSBs. Immunoblotting for both molecules in PE and PTL placenta samples and in trophoblast cells validated a connection. Co-immunoprecipitation studies revealed interactions between RAGE and pATM and MRE11 during DNA-DSBs. Reduced cellular invasion confirmed the role of genomic instability in trophoblastic function. Collectively, these experiments identified genomic instability in pregnancy complications, the impact of defective DNA on trophoblast function, and a possible RAGE-mediated mechanism during DNA-DSB repair.

CRISPR/Cas9-based precise excision of SlHyPRP1 domain(s) to obtain salt stress-tolerant tomato.

CRISPR/Cas9-based multiplexed editing of SlHyPRP1 resulted in precise deletions of its functional motif(s), thereby resulting in salt stress-tolerant events in cultivated tomato. Crop genetic improvement to address environmental stresses for sustainable food production has been in high demand, especially given the current situation of global climate changes and reduction of the global food production rate/population rate. Recently, the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based targeted mutagenesis has provided a revolutionary approach to crop improvement. The major application of CRISPR/Cas in plant genome editing has been the generation of indel mutations via error-prone nonhomologous end joining (NHEJ) repair of DNA DSBs. In this study, we examined the power of the CRISPR/Cas9-based novel approach in the precise manipulation of protein domains of tomato hybrid proline-rich protein 1 (HyPRP1), which is a negative regulator of salt stress responses. We revealed that the precise elimination of SlHyPRP1 negative-response domain(s) led to high salinity tolerance at the germination and vegetative stages in our experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multidomain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.

Second-Generation Antiandrogen Therapy Radiosensitizes Prostate Cancer Regardless of Castration State through Inhibition of DNA Double Strand Break Repair.

Simple SummaryThe combination of RT and the first generation AR blockers to improve the outcome in prostate cancer remain a matter of controversial debate in clinical trials. In the current study we aim to investigate the effect of three FDA approved second-generation antiandrogens (abiraterone acetate, apalutamide and enzalutamide), as more potent inhibitors of the AR signaling, on the cytotoxicity of RT in pre-clinical models. In vitro and ex vivo analyses revealed a strong radiosensitising effect for the second-generation antiandrogens, regardless of the castration state. The first-generation AR-blocker bicalutamide failed to show any radiosensitising effect. The radiosensitising effect of the second-generation antiandrogens was attributed to the inhibition of DSB repair. Together, we provide a proof-of-principle pre-clinical evidence to rationalize the clinical use of the second-generation antiandrogens to enhance the effect of IR as a potential strategy to improve the outcomes of PCa patients with localized disease who undergo ablative RT.(1) Background: The combination of the first-generation antiandrogens and radiotherapy (RT) has been studied extensively in the clinical setting of prostate cancer (PCa). Here, we evaluated the potential radiosensitizing effect of the second-generation antiandrogens abiraterone acetate, apalutamide and enzalutamide. (2) Methods: Cell proliferation and agarose-colony forming assay were used to measure the effect on survival. Double strand break repair efficiency was monitored using immunofluorescence staining of γH2AX/53BP1. (3) Results: We report retrospectively a minor benefit for PCa patients received first-generation androgen blockers and RT compared to patients treated with RT alone. Combining either of the second-generation antiandrogens and 2Gy suppressed cell growth and increased doubling time significantly more than 2Gy alone, in both hormone-responsive LNCaP and castration-resistant C4-2B cells. These findings were recapitulated in resistant sub-clones to (i) hormone ablation (LNCaP-abl), (ii) abiraterone acetate (LNCaP-abi), (iii) apalutamide (LNCaP-ARN509), (iv) enzalutamide (C4-2B-ENZA), and in castration-resistant 22-RV1 cells. This radiosensitization effect was not observable using the first-generation antiandrogen bicalutamide. Inhibition of DNA DSB repair was found to contribute to the radiosensitization effect of second-generation antiandrogens, as demonstrated by a significant increase in residual γH2AX and 53BP1 foci numbers at 24h post-IR. DSB repair inhibition was further demonstrated in 22 patient-derived tumor slice cultures treated with abiraterone acetate before ex-vivo irradiation with 2Gy. (4) Conclusion: Together, these data show that second-generation antiandrogens can enhance radiosensitivity in PCa through DSB repair inhibition, regardless of their hormonal status. Translated into clinical practice, our results may help to find additional strategies to improve the effectiveness of RT in localized PCa, paving the way for a clinical trial.

Genome-wide identification of DNA double-strand break repair genes and transcriptional modulation in response to benzo[α]pyrene in the monogonont rotifer Brachionus spp.

The DNA repair system has evolved from the common ancestor of all life forms and its function is highly conserved within eukaryotes. In this study, to reveal the role of DNA double-strand break repair (DSB) genes in response to benzo[α]pyrene (B[α]P), we first identified DSB genes in relation to homologous recombination and non-homologous end joining events in four Brachionus rotifer spp.: B. calyciflorus, B. koreanus, B. plicatilis, and B. rotundiformis. In all the Brachionus spp., 39 orthologous genes to human DSB repair genes were identified. Furthermore, three genes in B. koreanus, two genes in B. plicatilis, and one gene in B. calyciflorus and B. rotundiformis were present as duplicated genes, indicating that these genes were diversified over speciation in the genus Brachionus. Moreover, we compared DSB repair genes on the gene structures in four monogonont Brachionus rotifers and the bdelloid rotifer Adineta vaga, which possesses highly efficient DNA repair ability. The transcriptional responses of four monogonont Brachionus rotifers in response to B[α]P exposure showed how B[α]P exposure led to DSBs and subsequently recruited DNA DSB repair pathways in the rotifer B. koreanus. Taken together, this study provides a better understanding of the potential role of DSB repair genes in the monogonont rotifer Brachionus spp. in response to B[α]P.

Abstract 6164: Small-molecule targeting of the NPM1/Rad51 repair pathway for the radiosensitization of cancer

Abstract Nucleophosmin1 (NPM1) is rapidly recruited to sites of DNA double-strand breaks and is required for radiation-induced Rad51 foci formation. Inhibition of NPM1 or Rad51 is a radiosensitizing event. YTR107 is a small molecule, identified in a forward chemical genetics functional structure/activity screening, that binds to the amino terminus of NPM1 and prevents NPM1 recruitment to damaged chromatin. In silico docking studies of YTR107 and the crystal structure of the NPM1 N-terminal oligomerization domain revealed that YTR107 docked to monomeric NPM1 in a pocket crucial for forming an interface between neighboring subunits of the NPM1 pentamer. YTR107 inhibited radiation-induced Rad51 foci formation and repair of DNA DSBs, as measured by a neutral comet assay. Colony formation assays revealed that YTR107 radiosensitized 7 NSCLC lines, HT29 colorectal cancer cells, D54 glioblastoma cells, PANC1 pancreatic cancer cells, and 2 breast cancer cell lines (dose modifying factors of > 1.5; cells irradiated with 300 kVp/10mA X-rays @ 2 Gy/min). Radiosensitization was found to be NPM1-dependent as YTR107 did not radiosensitize an NPM1 null mouse embryo fibroblast cell line. YTR107 produced a 4.7 fold increase in HT29 xenograft growth delay (time required for the tumor to increase 4-fold in volume (p = 0.001) following 10 mg/kg YTR107 administered 30 min prior to administering 3 Gy to the tumor for 7 consecutive days). YTR107 increased the survival of A549 tumor-bearing mice determined 70 days after the last treatment. Survival of tumor-bearing mice was 60% for mice treated with YTR107 and 3 Gy vs 20% for mice treated with 3 Gy alone, p = 0.0012 (0 or 20 mg/kg YTR107 administered 1 hr prior to 0 or 2.2 Gy being administered to the tumor for 7 consecutive days). Female C57BL/6J mice were administered 0 or 10 mg/kg i.p. YTR107 for 5 consecutive days. Thirty five days after injection mice were euthanized and subjected to necropsy by a veterinarian trained in veterinary pathology. Gross and histological examination of liver, lung, thymus, heart, spleen, cerebellum, pancreas, small intestine, kidney, and adrenal gland did not reveal evidence of toxicity. Both a complete blood count and a white blood cell differential count were performed. No significant differences were noted. Mice were subjected to five daily injections of 30 mg/kg YTR107. This dose level was also tolerated without overt toxicity after 35 days. Mice were injected with 0 or 30 mg/kg YTR107 (i.p.) and 30 min later administered 0 or 16-Gy to the thorax. Pulmonary fibrotic lesions were quantified using respiratory-gated micro CT 136 days later. YTR107 did not increase the severity of radiation-induced lung fibrosis and did not affect body weights or life span of unirradiated or irradiated mice (p > 0.05) over the 136-day study. Thus, this study provides support for development of YTR107 as a therapeutic radiation sensitizer and was supported in part by R44CA228756-02 Citation Format: Konjeti R. Sekhar, Geri Traver, Peter A. Crooks, Michael L. Freeman. Small-molecule targeting of the NPM1/Rad51 repair pathway for the radiosensitization of cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6164.

Short-term treatment with imetelstat sensitizes hematopoietic malignant cells to a genotoxic agent via suppression of the telomerase-mediated DNA repair process

Imetelstat is a specific and competitive inhibitor of telomerase enzymatic activity. We demonstrated that imetelstat could interfere with the DNA repair process and enhance the effect of DNA damaging agents using hematological tumor cell lines. Short-term administration of imetelstat enhanced growth suppression by anticancer agents and radiation. It also upregulated γH2AX expression induced by irradiation. Immunofluorescence staining showed that both human telomerase reverse transcriptase (hTERT) and γH2AX were upregulated and co-localized in the nucleus of peripheral blood mononuclear cells after irradiation, suggesting that hTERT was involved in the DNA-DSB repair process. Imetelstat enhanced growth inhibitory effect of cytotoxic agents in short-term culture without shortening of telomeres, indicating that this effect was attributed by telomere length independent mechanism. Our results suggest that the combination of short-term treatment with imetelstat and cytotoxic agent is a promising strategy to treat a wide variety of hematopoietic malignancies.

The Role of Telomerase in Radiation-Induced Genomic Instability.

Findings from previous studies have suggested that the telomerase system is involved in radiation-induced genomic instability. In this study, we investigated the involvement of telomerase in the development and processing of chromosomal damage at different cell cycle stages after irradiation of human fibroblasts. Several response criteria were investigated, including cell survival, chromosomal damage (using the micronucleus assay), G2-induced chromatid aberrations (using the conventional G2 assay as well as a chemically-induced premature chromosome condensation assay) and DNA double-strand breaks (DSBs; using γ-H2AX, 53BP1 and Rad51) in an isogenic pair of cell lines: BJ human foreskin fibroblasts and BJ1-hTERT, a telomerase-immortalized BJ cell line. To distinguish among G1, S and G2 phase, cells were co-immunostained for CENP-F and cyclin A, which are tightly regulated proteins in the cell cycle. After X-ray irradiation at doses in the range of 0.1-6 Gy, the results showed that for cell survival and micronuclei induction, where the overall effect is dominated by the cells in G1 and S phase, no difference was observed between the two cell types; in contrast, when radiation sensitivity at the G2 stage of the cell cycle was analyzed, a significantly higher number of chromatid-type aberrations (breaks and exchanges), and higher levels of γ-H2AX and of Rad51 foci were observed for the BJ cells compared to the BJ1-hTERT cells. Therefore, it can be concluded that telomerase appears to be involved in DNA DSB repair processes, mainly in the G2 phase. These data, taken overall, reinforce the notion that hTERT or other elements of the telomere/telomerase system may defend chromosome integrity in human fibroblasts by promoting repair in G2 phase of the cell cycle.

HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells

(Molecular Cell 77, 384–394.e1–e4; January 16, 2020) The authors report two minor errors in Figure 3G of the originally published article. In this panel, there appeared a letter “E” that was not relevant to the figure and may have been confusing to readers. Also, in the same panel, the authors report having inadvertently copied an older version of the bar graph, with fewer replicates, which is different from the one provided to reviewers in the revised version of the manuscript. These errors were made during the generation of high-resolution images toward the end of the production process and have since been corrected online. The errors do not change the message of the figure, nor do they impact the overall conclusions of the article. The authors apologize for any inconvenience.Figure 3GHMCES Deficiency Leads to a Defect in CSR in the CH12 B Cell Line (original)View Large Image Figure ViewerDownload Hi-res image Download (PPT) HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B CellsShukla et al.Molecular CellDecember 2, 2019In BriefHMCES (5hmC binding, embryonic stem cell-specific protein), originally found to bind 5-hydroxymethylcytosine (5hmC), was recently reported to covalently crosslink to DNA at abasic sites. Shukla et al. show that HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells. Full-Text PDF Open Archive

Characterization of t-loop formation by TRF2

ABSTRACT T-loops are thought to hide telomeres from DNA damage signaling and DSB repair pathways. T-loop formation requires the shelterin component TRF2, which represses ATM signaling and NHEJ. Here we establish that TRF2 alone, in the absence of other shelterin proteins can form t-loops. Mouse and human cells contain two isoforms of TRF2, one of which is uncharacterized. We show that both isoforms protect telomeres and form t-loops. The isoforms are not cell cycle regulated and t-loops are present in G1, S, and G2. Using the DNA wrapping deficient TRF2 Topless mutant, we confirm its inability to form t-loops and repress ATM. However, since the mutant is also defective in repression of NHEJ and telomeric localization, the role of topological changes in telomere protection remains unclear. Finally, we show that Rad51 does not affect t-loop frequencies or telomere protection. Therefore, alternative models for how TRF2 forms t-loops should be explored.

Effectiveness of the DNA double-strand breaks repair system in lymphocytes of patients with cognitive impairments and healthy volunteers

The individual differences in the efficiency of DNA DSB repair were estimated by the level of residual γH2AX foci after γ-irradiation at a dose of 2 Gy, in lymphocytes of patients with amnestic mild cognitive impairment (AMCI) and Alzheimer's disease (AD) and of healthy volunteers. Lymphocytes were isolated from the peripheral blood of the examined patients and were frozen in a medium for freezing cells. Before the study, the lymphocytes were thawed, suspended in RPMI 1640 culture medium supplemented with 10% inactivated fetal bovine serum, and half of the cells were γ-irradiated at 4°C from a 60Co source on a GUT-200M facility at a dose of 2 Gy (a dose rate of 0.75 Gy/min). Control and irradiated lymphocytes were cultured for 24 h, collected, fixed, and stored until the study of the number of spontaneous and residual foci of γH2AX using fluorescent microscopy after staining with fluorescent labeled antibodies. In lymphocytes of patients with AMCI and AD a higher number of residual γH2AX foci in lymphocytes and the higher number of lymphocytes with foci were found compared with healthy volunteers. This indicates a decrease in the ability to repair DNA DSB in these patients. Indicators of cellular immunity and the concentration of TNF-α in the blood serum in the group of examined patients were normal. In the group of patients with the cognitive impairments (AMCI+AD), a correlation was found between the number of residual foci of γH2AX and the number of CD3+CD4+ lymphocytes and the concentration of proinflammatory cytokine TNF-α in the blood serum. This suggests the development of stronger neuroinflammation in patients with reduced ability to repair DNA DSB in this pathology.

HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells.

HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.

Imetelstat Sensitizes Hematopoietic Malignancy Cells to Genotoxic Agent Via Suppression of Telomerase Mediated DNA Repair Process

Telomerase is the ribonucleoprotein enzyme that has the main function of extension of telomeric repeat. Telomerase consists of two constructions: telomerase reverse transcriptase protein (hTERT) and RNA template. Inhibition of telomerase activity is considered to be a therapeutic approach for malignant tumors since telomerase is dominantly expressed in germ cells, stem cells, and cancer cells but not in normal cells. We found that (TTAGGG)n repeats could be integrated and repair artificially induced DNA double strand break (DSB) sites (Onozawa M et al. PNAS, 2014). Therefore, we hypothesized that inhibition of telomerase activity may interfere with the DNA repair process and enhance the effects of DNA damaging agents. Imetelstat competitively suppresses telomerase activity. It has a binding site complementary to the RNA template of telomerase and induces specific inhibition. Imetelstat showed clinical benefits in patients with essential thrombocytosis or myelofibrosis used as monotherapy. However, the effectiveness of imetelstat combined with other cytotoxic agent for hematological malignancies is not clear. We therefore evaluated the synergistic anti-tumor effects of imetelstat and cytotoxic agents on hematological malignancies. First, the expression of hTERT mRNA, protein, and activity was confirmed in 8 human cell lines of hematological malignancies (U937, HL60, PALL2, CEM, IM9, RPMI8226, ST1, and SU9T01) but not in peripheral blood mononuclear cells (PBMCs) from a healthy individual. There was a significant correlation between mRNA expression and activity measured by TRAP assay (r=0.839, 95% confidence interval: 0.396-0.965, p=0.00467). The expression of hTERT mRNA in clinical samples was analyzed using primary acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) cells. The expression level of hTERT in ALL cells was significantly higher than that in AML cells (p=0.0093). The telomerase inhibitor, imetelstat had a concentration-dependent effect on telomerase inhibition, while a control oligo did not affect telomerase activity. The expression of hTERT protein was not suppressed by imetelstat treatment. Imetelstat also had a concentration-dependent suppressive effect on cell growth without affecting cell viability. Next, we evaluated the synergistic anti-tumor effects of etoposide and imetelstat at the concentration at which it did not affect cell growth by monotherapy. Cell proliferation at day 4 was inhibited by coadministration of etoposide (1 µM) and imetelstat (1 µM) compared to that with administration of etoposide alone (Figure A). Western blot analysis showed that gamma-H2AX expression level after radiation and imetelstat administration was significantly higher than that of after radiation alone (Figure B). Therefore, imetelstat enhanced DNA-DSB induced by a genotoxic agent. We determined whether the synergistic effect was mediated by shortening of telomere length. Telomere length was analyzed in long-term culture using the flow-FISH method. Imetelstat significantly shortened telomere length compared to a control oligo after day 14, while there was almost no change in telomere length at day 4. We also analyzed the changes of hTERT activity and gamma-H2AX expression after radiation in PBMCs from a healthy individual. Although hTERT activity was not detected in stable state in PBMC, hTERT activity appeared and gradually increased over time and gamma-H2AX expression peaked from 2 to 4 hours after radiation. Images of immunofluorescence staining revealed co-localization of hTERT and gamma-H2AX in the nucleus of PBMCs after radiation (Figure C). Therefore, hTERT induced by DNA damage might be directly involved in the DNA-DSB repair process. We clearly showed synergistic effects of imetelstat and a genotoxic agent in short-term treatment, in which shortening of telomeres was not observed. Potentially, telomerase inhibition could stand as a beneficial addition to other treatment methods when the inhibitor is administered at low non-toxic doses. Our hypothesis needs to be clarified in a clinical trial. Further studies are required to optimize these therapies in a variety of hematopoietic malignancies or other cancers. Figure Disclosures Nakagawa: akeda Pharmaceutical Company Limited: Research Funding. Teshima:Novartis: Honoraria, Research Funding.

DNA-PKcs is activated under nutrient starvation and activates Akt, MST1, FoxO3a, and NDR1

BackgroundPresence of unperfused regions containing cells under hypoxia and nutrient starvation; contributes to radioresistance in solid human tumors. We have previously reported that cultured cells; under nutrient starvation show resistance to ionizing radiation compare with cells under normal; condition, and that nutrient starvation increases ATM activity, which causes cellular resistance to; ionizing radiation (Murata et al., BBRC2018). For further investigation of molecular mechanisms; underlying radioresistance of cells under nutrient starvation, effects of nutrient starvation on activity; of DNA-PKcs have been investigated because both DNA-PKcs and ATM belong to the PIKK family; and are required for DNA DSBs repair. In addition to DNA-PKcs, effects of nutrient starvation on; activities of FoxO3a and its regulators Akt, MST1 and AMPK have been investigated because FoxO3a; mediates cellular responses to stress and is activated under nutrient starvation. MethodsA human glioblastoma cell line, T98G was used to examine the effects of nutrient starvation on activities and expression of DNA-PKcs, Akt, MST1, FoxO3a, NDR1, and AMPK. To elucidate; signal transduction pathways for FoxO3a activation under nutrient starvation, we examined effects of; specific inhibitors or siRNA for DNA-PKcs or Akt on activities and expression of MST1, FoxO3, NDR1, andAMPK. ResultsUnder nutrient starvation, phosphorylations of DNA-PKcs at Ser2056, Akt at Ser473, MST at Thr183, FoxO3a at Ser413, NDR1 at Ser281 and Thr282, and AMPK at Thr172 were increased, which suggests their activation. Nutrient starvation did not affect expression of DNA-PKcs, Akt, MST1, or NDR1, with decreased expression of FoxO3a and increased expression of AMPK. Inhibition; of DNA-PK suppressed phosphorylation of Akt under nutrient starvation. Inhibition of DNA-PK or; Akt suppressed phosphorylations of MST1, FoxO3a, and NDR1 under nutrient starvation, which; suggests DNA-PKcs and Akt activate MST1, FoxO3a, and NDR1. Inhibition of DNA-PK did not; suppress phosphorylation ofAMPK under nutrient starvation. ConclusionOur data suggest that DN-PKcs is activated under nutrient starvation and activates AktMST1, FoxO3a, and NDR1.

Induction of p73, Δ133p53, Δ160p53, pAKT lead to neuroprotection via DNA repair by 5-LOX inhibition

The inflammatory process plays a key role in neurodegenerative disorder. The inflammatory molecule, 5-lipooxygenase (5-LOX), protein is involved in the pathologic phenotype of AD which includes Aβ amyloid deposition and tau hyperphosphorylation. This study aims to identify the mechanistic role in neuroprotection by 5-LOX inhibitor in neurotoxic SH-SY5Y cell line model by evaluating different cell survival pathway. The neurotoxic SH-SY5Y cells were developed by the treatment of Aβ25-35. The cells were then treated with 5-LOX peptide inhibitor, YWCS to prevent neurotoxicity reported earlier from our lab. The effect of 5-LOX inhibition on cell survival pathways were determined by western blot experiment with different doses of peptide by using polyclonal anti body of p53, anti-Akt and anti-phosphorylated Akt. Immunoprecipitation and mass spectroscopic studies were done to identify the altered proteins appeared on the blot. Over expression of phosphorylated Akt and 3 bands on p53 lane blot other than p53 were observed. Three bands were identified as isoforms of p53 which correspond to p73, Δ133p53 and Δ160p53 in the cells treated only with 80µM of YWCS compare to untreated cells. However, no alteration of total p53 and Akt were observed in treated cells. The results exposed the novel mechanistic pathway of neuroprotection by 5-LOX inhibition is likely to be mediated by DNA DSB repair through p53 isoforms and PI3K/Akt pathway. Our finding has opened a new window in the therapeutic approach for the prevention of AD.

Exploiting Conditional Vulnerabilities Caused by the Systems Level Effects of Tumor Treating Fields

Tumor Treating Fields (TTFields) delivers low-intensity, intermediate frequency, alternating electric fields non-invasively to a tumor. Optune, a TTFields delivery device, has been approved for glioblastoma and clinical trials are ongoing for other cancers. Other mechanisms of action besides interference with mitosis have been identified and suggest alternative clinical strategies to the use of TTFields. Human NSCLC and pancreatic cell lines were used to examine TTFields effects in vitro while an tumor explant system was used for ex vivo studies. The Inovitro system was used to generate TTFields. Quantification of DNA damage was done by using 53BP1 and γ-H2AX foci. The DNA fiber assay was used to quantify newly synthesized DNA to examine replication fork dynamics. R loop detection was done by dot-blot and immunofluorescence using the S9.6 antibody. Clonogenic cell survival was performed with TTFields combined with specific agents (ionizing radiation (IR), cisplatin, olaparib, IR plus cisplatin, IR plus olaparib). We have developed an ex vivo model for lung tumors that are exposed to to TTFields. TTFields exposure decreased the expression of FANC/BRCA1 pathway genes resulting in the downregulation of DNA DSB repair of IR-induced DSBs. Most importantly, TTFields alone increased the number of γH2AX foci and the incidence of chromatid aberrations as a function of TTFields exposure time. Furthermore, TTFields exposure decreased the length of newly replicated DNA and increase R-loop formation as a function of exposure time, suggesting that TTFields induce replication stress. We hypothesized that by applying TTFields, a systems level conditional vulnerability develops (inhibition of DNA repair, increased replication stress, production of DSBs, reduced mitophagy, and other stresses) rendering cells more susceptible to agents that cause DNA damage or replication stress or interfere with its repair. In agreement with our hypothesis, NSCLC cell susceptibility to radiation increased when cells were exposed to TTFields prior to IR treatment compared to IR treatment followed by TTFields. TTFields also enhanced cisplatin toxicity in a synergistic manner. Furthermore, TTFields exposure of NSCLC and pancreatic tumor cells concomitant with the PARP inhibitor Olaparib followed by radiation resulted in synergistic cell killing compared to radiation or Olaparib alone or in combination. Our initial results of ex vivo tumor explants exposed to TTFields supports our in vitro results in that we see the down-regulation of key DNA repair pathway proteins as was seen in our in vitro studies. Our data identifies systems level perturbations to key molecular and biochemical pathways that render tumor cells vulnerable to a number of different agents. We suggest that TTFields would serve well as neoadjuvant therapy prior to radiation treatment and either prior to or concomitant with chemotherapy agents.

Maintenance BEZ235 Treatment Prolongs the Therapeutic Effect of the Combination of BEZ235 and Radiotherapy for Colorectal Cancer.

Our previous study demonstrated that administration of NVP-BEZ235 (BEZ235), a dual PI3K/mTOR inhibitor, before radiotherapy (RT) enhanced the radiotherapeutic effect in colorectal cancer (CRC) cells both in vitro and in vivo. Here, we evaluated whether maintenance BEZ235 treatment, after combinatorial BEZ235 + RT therapy, prolonged the antitumor effect in CRC. K-RAS mutant CRC cells (HCT116 and SW480), wild-type CRC cells (HT29), and HCT116 xenograft tumors were separated into the following six study groups: (1) untreated (control); (2) RT alone; (3) BEZ235 alone; (4) RT + BEZ235; (5) maintenance BEZ235 following RT + BEZ235 (RT + BEZ235 + mBEZ235); and (6) maintenance BEZ235 following BEZ235 (BEZ235 + mBEZ235). RT + BEZ235 + mBEZ235 treatment significantly inhibited cell viability and increased apoptosis in three CRC cell lines compared to the other five treatments in vitro. In the HCT116 xenograft tumor model, RT + BEZ235 + mBEZ235 treatment significantly reduced the tumor size when compared to the other five treatments. Furthermore, the expression of mTOR signaling molecules (p-rpS6 and p-eIF4E), DNA double-strand break (DSB) repair-related molecules (p-ATM and p-DNA-PKcs), and angiogenesis-related molecules (VEGF-A and HIF-1α) was significantly downregulated after RT + BEZ235 + mBEZ235 treatment both in vitro and in vivo when compared to the RT + BEZ235, RT, BEZ235, BEZ235 + mBEZ235, and control treatments. Cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), 53BP1, and γ-H2AX expression in the HCT116 xenograft tissue and three CRC cell lines were significantly upregulated after RT + BEZ235 + mBEZ235 treatment. Maintenance BEZ235 treatment in CRC cells prolonged the inhibition of cell viability, enhancement of apoptosis, attenuation of mTOR signaling, impairment of the DNA-DSB repair mechanism, and downregulation of angiogenesis that occurred due to concurrent BEZ235 and RT treatment.