Increased DNA Double-strand Breaks Research Articles

Activation of EGFR-DNA-PKcs pathway by IGFBP2 protects esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage

BackgroundThe incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett’s esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks.MethodsReal-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage.ResultsOur results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett’s cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure.ConclusionsIGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.

Decreased KAT5 Expression Impairs DNA Repair and Induces Altered DNA Methylation in Kidney Podocytes

Altered DNA methylation plays an important role inthe onset and progression of kidney disease. However, little is known about how the changes arise in disease states. Here, we report that KAT5-mediated DNA damage repair is essential for the maintenance of kidney podocytes and is associated with DNA methylation status. Podocyte-specific KAT5-knockout mice develop severe albuminuria with increased DNA double-strand breaks (DSBs), increased DNA methylation of thenephrin promoter region, and decreased nephrin expression. Podocyte KAT5 expression is decreased, whereas DNA DSBs and DNA methylation are increased in diabetic nephropathy; moreover, KAT5 restoration by gene transfer attenuates albuminuria. Furthermore, KAT5 decreases DNA DSBs and DNA methylation at the same nephrin promoter region, which indicates that KAT5-mediated DNA repair may be related to DNA methylation status. These results suggest a concept in which an environment of DNA damage repair, which occurs with decreased KAT5, may affect DNA methylation status.

HSP110 translocates to the nucleus upon genotoxic chemotherapy and promotes DNA repair in colorectal cancer cells.

A multicenter clinical study demonstrated the presence of a loss-of-function HSP110 mutation in about 15% of colorectal cancers, which resulted from an alternative splicing and was produced at the detriment of wild-type HSP110. Patients expressing low levels of wild-type HSP110 had excellent outcomes (i.e. response to an oxaliplatin-based chemotherapy). Here, we show in vitro, in vivo, and in patients' biopsies that HSP110 co-localizes with DNA damage (γ-H2AX). In colorectal cancer cells, HSP110 translocates into the nucleus upon treatment with genotoxic chemotherapy such as oxaliplatin. Furthermore, we show that HSP110 interacts with the Ku70/Ku80 heterodimer, an essential element of the non-homologous end joining (NHEJ) repair machinery. We also demonstrate by evaluating the resolved 53BP1 foci that depletion in HSP110 impairs repair steps of the NHEJ pathway, which is associated with an increase in DNA double-strand breaks and in the cells' sensitivity to oxaliplatin. HSP110-depleted cells sensitization to oxaliplatin-induced DNA damage is abolished upon re-expression of HSP110. Confirming a role for HSP110 in DNA non-homologous repair, SCR7 and NU7026, two inhibitors of the NHEJ pathway, circumvents HSP110-induced resistance to chemotherapy. In conclusion, HSP110 through its interaction with the Ku70/80 heterodimer may participate in DNA repair, thereby inducing a protection against genotoxic therapy.

Abstract 4158: Differential intestinal tumorigenesis and DNA repair response in APCMin/+ mice after acute and fractionated proton radiation

Abstract Ionizing radiation (IR) exposure is a risk factor for colorectal cancer (CRC) development. With increasing interest in exploring outer space, astronauts on prolonged space missions such as a mission to Mars are expected to receive IR doses that could heighten the risk of CRC. It is estimated that about 90% of the IR in outer space is energetic protons, and the radiation dose during solar particle events (SPEs) could reach up to 2 Gy behind shielding. However, the risk of CRC after energetic proton radiation is not yet fully defined due to lack of human or animal model data. The purpose of the current study was to quantitatively and qualitatively compare and characterize differences in intestinal tumorigenesis between acute and fractionated proton irradiation in APCMin/+ mice, a well-established mouse model for human CRC. Mice were exposed to 1.88 Gy of proton radiation delivered in a single fraction or in 4 equal daily fractions (0.47 Gy x 4). Since proton dose to astronauts are variable based on space environment, in this initial study we chose a high single dose and a lower daily dose pattern to assess intestinal tumorigenesis. Intestinal tumor frequency and grade were noted, and tumor samples were collected from irradiated and control mice euthanized 100 to 110 days after radiation exposure. Molecular analysis was focused on DNA damage and repair, and cellular proliferative signaling pathways relevant to CRC using immunoblots, immunohistochemistry, and qPCR. Relative to control and fractionated-protons, there was significantly higher intestinal tumor frequency and grade after acute (single dose) proton irradiation. Decreased cellular differentiation and increased oxidative damage to DNA was observed after acute proton radiation. At the molecular level, our data showed increased DNA double strand breaks associated with decreased levels of DNA repair proteins, and upregulation of proliferative β-catenin and Akt signaling after acute proton exposure. Since increased DNA damage was not associated with concomitant increased apoptosis, our data suggest continued proliferation of cells bearing sub-lethal damage to promote tumorigenesis. In the fractionated proton group, tumor frequency and grade as well as molecular changes were comparable to sham-irradiated control group. When considered along with decreased tumor frequency and upregulation of DNA repair pathways after fractionation, our data are suggestive of marked differences in carcinogenic effects of acute vs. fractionated proton radiation. In summary, our data suggest that acute exposure to proton radiation is associated with higher risk of intestinal tumorigenesis in APCMin/+ mice with implications for gastrointestinal cancer risk in astronauts undertaking long duration space missions and in patients undergoing proton radiotherapy. Citation Format: Kamal Datta, Shubhankar Suman, Santosh Kumar, Albert J. Fornace. Differential intestinal tumorigenesis and DNA repair response in APCMin/+ mice after acute and fractionated proton radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4158.

Abstract 2827: Alternative non-homologous end joining DNA repair as therapeutic target in multiple myeloma

Abstract Alternative Non Homologous End Joining DNA repair as therapeutic target in Multiple MyelomaDaniele Caracciolo, Martina Montesano, Emanuela Altomare, Grazia Consolo, Nicola Amodio, Maria Teresa Di Martino, Marco Rossi, Cirino Botta, Eugenio Morelli, Giada Juli, Pierosandro Tagliaferri, Pierfrancesco Tassone. UMG of Catanzaro, Catanzaro, ItalyBackground- Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability. We previously demonstrated that LIG3-dependent Alt-NHEJ repair is significantly involved in the genomic instability which promote survival and drug resistance of MM cells. Based on this rationale, we aimed to elucidate the efficacy of available Alt-NHEJ pathway inhibitors as new therapeutic agents in MM. Materials and methods- Cell proliferation and apoptosis were evaluated with CellTiter-Glo assay and Annexin V staining. Alt-NHEJ repair was evaluated using EJ2-GFP and EJ5-GFP plasmids. LIG3, LIG1, PARP1, Caspase 3 and p-H2AX, levels were analyzed by Western blot of whole protein extracts. In vivo anti-MM activity was evaluated in NOD-SCID mice bearing subcutaneous AMO-1 Bortezomib resistant (ABZB) xenografts, daily treated with Olaparib via oral gavage. Results- By interrogating public available datasets, we found that higher mRNA expression of Alt-NHEJ core components was correlated with shorter survival of MM patients. Indeed, plasmid-based reporter assays confirmed a prevalent activation of Alt-NHEJ repair in MM cells as compared to normal lymphoblastoid cell lines, except for U266 cells which instead displayed a prevalence of Canonical-NHEJ repair activity. Next, we attempted to evaluate the effects of available Alt-NHEJ inhibitors on MM cell survival. Consistently, L67 (LIG3-LIG1 inhibitor) or Olaparib (PARP-inhibitor) induced a significant reduction of proliferation and clonogenic cell growth of different MM cell lines at low micromolar concentrations, while no significant effects on survival were observed in U226 as well as in normal lymphoblastoid cells. Importantly L67 or Olaparib impaired viability of MM cell lines or primary plasmacells co-cultured on stromal cells, thus overcoming the supporting role played by MM-microenvironment on plasma-cells survival. As result of Alt-NHEJ repair inhibition, anti-proliferative effects were correlated to increase of DNA double-strand breaks (DSBs), cell cycle arrest and finally apoptosis. Notably, we observed that Bortezomib resistant AMO-1 derivative cells (ABZB) exhibited significantly increased sensitivity to a combination of L67 and Olaparib that increased the number of DSBs. Moreover, Alt-NHEJ inhibitors synergistically sensitized primary cells as well as MM cell lines to Bortezomib. Finally, to demonstrate the in vivo relevance of our findings, we showed that clinically available Parp-inhibitor Olaparib, exerted a significant anti-MM activity on ABZB cells injected in immunocompromised mice. Conclusion- Taken together, our findings indicate that MM cells are addicted to Alt-NHEJ repair parthway, which therefore represent a novel druggable target pathway in MM. Citation Format: Daniele Caracciolo, Martina Montesano, Emanuela Altomare, Grazia Consolo, Nicola Amodio, Maria Teresa Di Martino, Marco Rossi, Cirino Botta, Eugenio Morelli, Giada Juli, Pierosandro Tagliaferri, Pierfrancesco Tassone. Alternative non-homologous end joining DNA repair as therapeutic target in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2827.

Abstract 3750: Xenoestrogens cause estrogen receptor-dependent R-loop formation and DNA damage

Abstract Background: The hormone 17β-estradiol (E2) plays an important role in breast cell proliferation and development but prolonged exposures and higher levels of E2 have been linked to genomic instability and breast cancer. Recent studies show E2 induced transactivations lead to DNA damage. A variety of environmental chemicals mimic the activities of estrogen, referred to as xenoestrogens. Thus, our hypothesis is to determine whether xenoestrogens stimulate transcriptional response and induce DNA damage similarly like E2. Methods: The T-47D breast cancer cell line is used as a model because it expresses both estrogen receptors (ERα & ERβ) as found in normal breast tissue. Cells were exposed a physiologic range of E2 (17β-estradiol) [0.5-100nM] and the xenoestrogens, e.g., benzophenone-3 (BP-3) [0.5-50µM] and propyl paraben (PP) [0.5-10µM] with or without ER-antagonist ICI (1µM) for 24 hours. Transcriptional responses were quantified using an integrated ERE-luciferase reporter and qPCR for endogenous genes. Proliferation was monitored using the Alamar Blue assay. DNA damage was determined using γ-H2AX. R-loops were detected using the S9.6 antibody against DNA-RNA hybrids in the genome. Results: Exposure of T-47D cells to physiologic levels of E2, PP and BP3 causes significant increase in DNA double-strand breaks (DSBs) as determined by γ-H2AX staining. We demonstrate a concomitant increase in R-loop formation with increase in γH2AX intensity. Treatment with RNaseH depletes R-loop intensity, corroborating the result. E2 and PP induce transactivation as determined by Luciferase reporter assay as well as expression of ER-response genes (PGR and AREG), whereas BP3 has modest effect on transactivation of PGR. ER transactivation as well as R-loop formation by E2, PP and BP3 were abrogated with ICI, demonstrating ER-dependency of R-loop formation. Consistent with the transcriptional response, proliferation studies determined that E2 showed highest proliferation followed by PP. However, BP3 showed no increase in proliferation. Conclusions: Our results show that xenoestrogens have strikingly different activities in transactivation and proliferation. However, both xenoestrogens appear to stimulate R-loop accumulation that leads to DSB in an ER-dependent manner. Citation Format: Karen A. Dunphy, Prabin Dhangada Majhi, Aman Sharma, Amy L. Roberts, Elizabeth A. Daniele, Sallie S. Schneider, D Joseph Jerry. Xenoestrogens cause estrogen receptor-dependent R-loop formation and DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3750.

Enhancement of radiation effect on lung cancer A549 cells by gold nanoparticles.

e20505 Background: To study the in vitro radiosensitization effect of Thio-glucose capped gold nanoparticles(Glu-GNPs) combined with kilovolt or megavoltage X-ray on lung adenocarcinoma A549 cells. Methods: The crystal violet method was used to detect the cytotoxicity of Glu-GNPs on A549 cells and the radiosensitive inhibition of proliferation. The distribution of Glu-GNPs in the cells was observed by transmission electron microscopy (TEM). Dose-cell viability scores after irradiation with 160 KeV and 6 MeV X-rays, respectively, were determined by cell clone analysis and radio-biological parameters were calculated using a single-click multi-target model. The DNA double-strand breaks (Foci foci) of A549 cells were detected by γ-H2AX immunofluorescence. Results: TEM showed Glu-GNPs into A549 cells were aggregated state attached to intracellular membrane organelles - lysosomes or endosomes.Glu-GNPs at the concentration of 0-100nmol / L did not significantly inhibit the viability of A549 cells (P > 0.05).Cell cloning experiments showed that the SER of 100nmol / L Glu-GNPs combined with 160KV and 6 MeV irradiation was 1.41 and 1.15, respectively (P < 0.05). Glu-GNPs significantly increased DNA double-strand breaks in A549 cells induced by 160KV and 6 MeV X-rays (P < 0.05). Conclusions: Nano-gold can enter cells by endocytosis and locate in endosomes or lysosomes, and increase the sensitivity of lung adenocarcinoma A549 cells to radiation.

Elevated temperature increases meiotic crossover frequency via the interfering (Type I) pathway in Arabidopsis thaliana.

For most eukaryotes, sexual reproduction is a fundamental process that requires meiosis. In turn, meiosis typically depends on a reciprocal exchange of DNA between each pair of homologous chromosomes, known as a crossover (CO), to ensure proper chromosome segregation. The frequency and distribution of COs are regulated by intrinsic and extrinsic environmental factors, but much more is known about the molecular mechanisms governing the former compared to the latter. Here we show that elevated temperature induces meiotic hyper-recombination in Arabidopsis thaliana and we use genetic analysis with mutants in different recombination pathways to demonstrate that the extra COs are derived from the major Type I interference sensitive pathway. We also show that heat-induced COs are not the result of an increase in DNA double-strand breaks and that the hyper-recombinant phenotype is likely specific to thermal stress rather than a more generalized stress response. Taken together, these findings provide initial mechanistic insight into how environmental cues modulate plant meiotic recombination and may also offer practical applications.

Radiosensitization of clioquinol and zinc in human cancer cell lines

BackgroundWe previously reported that clioquinol acts as a zinc ionophore and inhibits the NF-κB signalling pathway. Other research has demonstrated that zinc deficiency plays a vital role in the occurrence and development of some solid tumours, and intracellular zinc supplementation may reverse this process and enhance the tumour sensitivity to anticancer treatment. Thus, we investigated the radiosensitization effects of clioquinol combined with zinc on HeLa and MCF-7 cells in vitro.MethodsThe dose effect of growth inhibition of clioquinol combined with zinc on cell viability was determined by a cell counting kit 8 (CCK-8) assay. The radiosensitization effect of clioquinol combined with zinc and/or MG132 in HeLa and MCF-7 cells was detected by the clonogenic assay. The cell cycle distribution and apoptosis of clioquinol combined with zinc on HeLa cells were analyzed by flow cytometry. A luciferase reporter construct was used to study the effect of clioquinol combined with zinc on NF-κB activity in HeLa cells. DNA double-strand breaks were detected by immunofluorescence. The mRNA and protein levels of ATM were analyzed by quantitative real-time PCR and Western blotting, respectively.ResultsOur research showed that clioquinol combined with zinc markedly increased the radiosensitivity of HeLa and MCF-7 cells in low toxic concentrations and resulted in a post-irradiation decrease in G2 phase arrest and an increase in apoptosis. Clioquinol combined with zinc also inhibited NF-κB activation, decreased ATM expression and increased DNA double-strand breaks (DSBs) induced by ionizing radiation.ConclusionsThese findings indicated that clioquinol combined with zinc enhanced the radiosensitivity of HeLa and MCF-7 cells by the down-regulation of ATM through the NF-κB signalling pathway.

Spliceosomal components protect embryonic neurons from R-loop-mediated DNA damage and apoptosis.

ABSTRACTRNA splicing factors are essential for the viability of all eukaryotic cells; however, in metazoans some cell types are exquisitely sensitive to disruption of splicing factors. Neuronal cells represent one such cell type, and defects in RNA splicing factors can lead to neurodegenerative diseases. The basis for this tissue selectivity is not well understood owing to difficulties in analyzing the consequences of splicing factor defects in whole-animal systems. Here, we use zebrafish mutants to show that loss of spliceosomal components, including splicing factor 3b, subunit 1 (sf3b1), causes increased DNA double-strand breaks and apoptosis in embryonic neurons. Moreover, these mutants show a concomitant accumulation of R-loops, which are non-canonical nucleic acid structures that promote genomic instability. Dampening R-loop formation by conditional induction of ribonuclease H1 in sf3b1 mutants reduced neuronal DNA damage and apoptosis. These findings show that splicing factor dysfunction leads to R-loop accumulation and DNA damage that sensitizes embryonic neurons to apoptosis. Our results suggest that diseases associated with splicing factor mutations could be susceptible to treatments that modulate R-loop levels.

Mlh1 is required for female fertility in Drosophila melanogaster: An outcome of effects on meiotic crossing over, ovarian follicles and egg activation

Mismatch repair (MMR) system, a conserved DNA repair pathway, plays crucial role in DNA recombination and is involved in gametogenesis. The impact of alterations in MMR family of proteins (bacterial MutS and MutL homologues) on mammalian fertility is well documented. However, an insight to the role of MMR in reproduction of non-mammalian organisms is limited. Hence, in the present study, we analysed the impact of mlh1 (a MutL homologue) on meiotic crossing over/recombination and fertility in a genetically tractable model, Drosophila melanogaster. Using mlh1e00130 hypomorphic allele, we report female specific adverse reproductive outcome for reduced mlh1 in Drosophila: mlh1e00130 homozygous females had severely reduced fertility while males were fertile. Further, mlh1e00130 females contained small ovaries with large number of early stages as well as significantly reduced mature oocytes, and laid fewer eggs, indicating discrepancies in egg production and ovulation. These observations contrast the sex independent and/or male specific sterility and normal follicular development as well as ovulation reported so far for MMR family proteins in mammals. However, analogous to the role(s) of mlh1 in meiotic crossing over and DNA repair processes underlying mammalian fertility, ovarian follicles from mlh1e00130 females contained significantly increased DNA double strand breaks (DSBs) and reduced synaptonemal complex foci. In addition, large proportion of fertilized eggs display discrepancies in egg activation and fail to proceed beyond stage 5 of embryogenesis. Hence, reduction of the Mlh1 protein level leads to defective oocytes that fail to complete embryogenesis after fertilization thereby reducing female fertility.

Радиосенсибилизация опухолевых стволовых клеток аденокарциномы молочной железы человека линии MCF-7 с помощью никлозамида

Purpose: To investigate the effect of niclosamide on the radiosensitivity of MCF-7 cancer stem cells (CSC) and to explore the molecular mechanism of radiosensitization.&#x0D; Material and methods: Cancer stem cells were obtained from MCF-7 mammary adenocarcinoma monolayer culture (2D) and propagated as mammospheres (3D) in serum-free DMEM/F12 supplemented with EGF, bFGF, B 27, heparin, and insulin in ultra-low attachment plates. The single cell suspensions were irradiated with 1, 2, 4, 6, and 8 Gy using 60Co source at a dose rate of 0.54 Gy/min. Controls were sham irradiated. The size of the CD44+/CD24-/low CSC fraction was measured using flow cytometry at the 7th and 14th days after irradiation. The radiosensitizing effect of niclosamide on the 2D- and 3D-cell cultures was assessed by the number of cells at the 7th and 14th days after irradiation with doses of 2 or 4 Gy. Niclosamide was added to the cell cultures in a dose of 2 µM 1 h before irradiation. Media with niclosamide were changed to corresponding media without niclosamide 24 h post-irradiation. Cell survival was evaluated by hemocytometer cell counting using trypan blue. The size of the CSC fraction was measured at the 7th day after the irradiation with or without niclosamide. To investigate the effect of niclosamide on DNA damage the level of histone γH2AX was measured by flow cytometry at 60 min after irradiation.&#x0D; Results: Culturing adherent MCF-7 cells line under non-adherent mammosphere-forming conditions led to significantly increase in CSCs proportion. CD44+/CD24–/low cell fraction in 2D-culture was 0.2 ± 0.1 %, and it was 3.2 ± 0.6 % in 3D-culture (the third-generation spheres). Subpopulation of CSCs increased with the increase in irradiation dose. At day 14 after irradiation with the dose of 8 Gy, CD44+/CD24–/low cell fraction in 2D-culture was 2.2 ± 0.6 %, and it was 12.0 ± 0.9 % in 3D-culture. Cells grown in mammosphere-culture were found to have a higher survival rate after irradiation at doses of 2 and 4 Gy than cells grown in adherent conditions. At day 7 after irradiation with 2 and 4 Gy the 3D-culture cells counts decreased less than the 2D-culture cells counts (92 ± 13 % and 61 ± 10 % versus 58 ± 4 % and 37 ± 3 % compared to non-irradiated control). Moreover, cells grown in mammosphere-culture showed a higher renewal capacity. At day 14 after irradiation with 2 and 4 Gy the number of 3D-culture cells was 66 ± 11 % and 30 ± 7 % while the number of adherent cells was 43 ± 2 % and 7 ± 1 % compared to non-irradiated control. It was found that niclosomide has radiosensitizing effect on cells grown in mammospheres. Pretreatment with niclosamide 1 h prior to irradiation resulted in decrease in cell survival. At day 7 after irradiation at 2 and 4 Gy in the presence of niclosamide, the numbers of 3D-culture cells were reduced by 13 % and 7 % compared to their numbers after irradiation with appropriate doses in the absence of niclosamide. Furthermore, niclosamide was shown to reduce the percentage of CSCs. At day 7 after irradiation with 2 and 4 Gy, 3D-culture showed elevated percentages of CD44+/CD24−/low cells: CSC fraction in mammospheres were 4.6 ± 0.3 % and 6.1 ± 0.5 %, respectively, and after pretreatment by niclosamide and irradiation the sizes of the CSC fractions were 2.5 ± 0.1 % and 4.0 ± 0.3 %, respectively. Radiosensitizing effect of niclosamide lasted for 2 weeks. At day 14 after irradiation at 2 and 4 Gy in combination with niclosamide the numbers of 3D-culture cells were reduced by 13 % and 9 % compared to cell numbers after irradiation with appropriate doses without of niclosamide. Radiosensitization of cells grown in mammospheres might be related with increase in DNA double-strand breaks (DSB) prodused by niclosomide. At 1 h after 2 and 4 Gy irradiation cells grown in mammospheres shown 2.4-fold and 4.8-fold increase in DNA DSB, as indicated by γH2AX levels, compared to baseline. The combined treatment of niclosamide and irradiation with 2 and 4 Gy resulted in 3.4-fold and 5.5-fold increase in the levels of histone γ-H2AX, respectively.&#x0D; Conclusion: Niclosamide increased the radiosensetivity not only of the bulk cells of mammary adenocarcinoma line MCF-7 but also of CSCs. The radiosensitizing effect of niclosamide was associated with increase in the level of DNA damage.

Impact of genotypic and phenotypic differences in sarcoma models on the outcome of photochemical internalization (PCI) of bleomycin

The low curative response to current treatment regimens for most soft tissue sarcomas indicates a strong need for alternative treatment strategies and predictive markers for treatment outcome. PCI (photochemical internalization) is a novel treatment strategy to translocate drugs into cytosol that otherwise would have been degraded in lysosomes. Two highly geno-and phenotypically different uterine and vulvar leiomyosarcoma cell lines, MES-SA and SK-LMS-1, were treated with bleomycin (BLM) activated by PCI (PCIBLM). The MES-SA cells were much more sensitive to PCIBLM than the SK-LMS-1 cells and the treatment induced a 7–8 fold higher increase in DNA double-strand breaks at the same dose of light as measure by γH2AX staining. A 3-fold higher induction of apoptosis and stronger activation of Bax and p21 was also measured in the P53WT MES-SA cells, compared to the P53mut SK-LMS-1 cells. The basal formation of reactive oxygen species (ROS) was 3-fold higher in SK-LMS-1 cells than in the MES-SA cells and SK-LMS-1 cells expressed glutathione peroxidase 1 (GPx1) and more superoxide dismutase 2 (SOD2) than the MES-SA cells. Glutathione depletion with the glutathione synthetase inhibitor buthionine sulfoximine increased the cytotoxic effect of the photochemical treatment (PDT) most strongly in the SK-LMS-1 cells, and reduced PCIBLM-induced H2AX activation in the MES-SA cells, but not in the SK-LMS-1 cells. The results indicate PCIBLM as a potential novel treatment strategy for soft tissue sarcomas, with antioxidant enzymes, in particular GPx1, and the P53 status as potential predictive markers for response to PCIBLM.

Drug-Repositioning Screens Identify Triamterene as a Selective Drug for the Treatment of DNA Mismatch Repair Deficient Cells

Purpose: The DNA mismatch repair (MMR) pathway is required for the maintenance of genome stability. Unsurprisingly, mutations in MMR genes occur in a wide range of different cancers. Studies thus far have largely focused on specific tumor types or MMR mutations; however, it is becoming increasingly clear that a therapy targeting MMR deficiency in general would be clinically very beneficial.Experimental Design: Based on a drug-repositioning approach, we screened a large panel of cell lines with various MMR deficiencies from a range of different tumor types with a compound drug library of previously approved drugs. We have identified the potassium-sparing diuretic drug triamterene, as a novel sensitizing agent in MMR-deficient tumor cells, in vitro and in vivoResults: The selective tumor cell cytotoxicity of triamterene occurs through its antifolate activity and depends on the activity of the folate synthesis enzyme thymidylate synthase. Triamterene leads to a thymidylate synthase-dependent differential increase in reactive oxygen species in MMR-deficient cells, ultimately resulting in an increase in DNA double-strand breaks.Conclusions: Conclusively, our data reveal a new drug repurposing and novel therapeutic strategy that has potential for the treatment of MMR deficiency in a range of different tumor types and could significantly improve patient survival. Clin Cancer Res; 23(11); 2880-90. ©2016 AACR.

Identification of a novel ATM inhibitor with cancer cell specific radiosensitization activity.

Treatment of advanced head and neck squamous cell carcinoma (HNSCC) is plagued by low survival and high recurrence rates, despite multimodal therapies. Presently, cisplatin or cetuximab is used in combination with radiotherapy which has resulted in minor survival benefits but increased severe toxicities relative to RT alone. This underscores the urgent need for improved tumor-specific radiosensitizers for better control with lower toxicities. In a small molecule screen targeting kinases, performed on three HNSCC cell lines, we identified GSK635416A as a novel radiosensitizer. The extent of radiosensitization by GSK635416A outperformed the radiosensitization observed with cisplatin and cetuximab in our models, while exhibiting virtually no cytotoxicity in the absence of radiation and in normal fibroblast cells. Radiation induced phosphorylation of ATM was inhibited by GSK635416A. GSK63541A increased DNA double strand breaks after radiation and GSK63541A mediated radiosensitization was lacking in ATM-mutated cells thereby further supporting the ATM inhibiting properties of GSK63541A. As a novel ATM inhibitor with highly selective radiosensitizing activity, GSK635416A holds promise as a lead in the development of drugs active in potentiating radiotherapy for HNSCC and other cancer types.

DNA double-strand breaks and Aurora B mislocalization induced by exposure of early mitotic cells to H2O2 appear to increase chromatin bridges and resultant cytokinesis failure

Aneuploidy, an abnormal number of chromosomes that is a hallmark of cancer cells, can arise from tetraploid/binucleated cells through a failure of cytokinesis. Reactive oxygen species (ROS) have been implicated in various diseases, including cancer. However, the nature and role of ROS in cytokinesis progression and related mechanisms has not been clearly elucidated. Here, using time-lapse analysis of asynchronously growing cells and immunocytochemical analyses of synchronized cells, we found that hydrogen peroxide (H2O2) treatment at early mitosis (primarily prometaphase) significantly induced cytokinesis failure. Cytokinesis failure and the resultant formation of binucleated cells containing nucleoplasmic bridges (NPBs) seemed to be caused by increases in DNA double-strand breaks (DSBs) and subsequent unresolved chromatin bridges. We further found that H2O2 induced mislocalization of Aurora B during mitosis. All of these effects were attenuated by pretreatment with N-acetyl-L-cysteine (NAC) or overexpression of Catalase. Surprisingly, the PARP inhibitor PJ34 also reduced H2O2-induced Aurora B mislocalization and binucleated cell formation. Results of parallel experiments with etoposide, a topoisomerase IIα inhibitor that triggers DNA DSBs, suggested that both DNA DSBs and Aurora B mislocalization contribute to chromatin bridge formation. Aurora B mislocalization also appeared to weaken the “abscission checkpoint”. Finally, we showed that KRAS–induced binucleated cell formation appeared to be also H2O2-dependent. In conclusion, we propose that a ROS, mainly H2O2 increases binucleation through unresolved chromatin bridges caused by DNA damage and mislocalization of Aurora B, the latter of which appears to augment the effect of DNA damage on chromatin bridge formation.

Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell-like spheroids, and tumor xenografts through multiple cell signaling pathways.

OBJECTIVE Defects in the apoptotic machinery and augmented survival signals contribute to drug resistance in glioblastoma (GBM). Moreover, another complexity related to GBM treatment is the concept that GBM development and recurrence may arise from the expression of GBM stem cells (GSCs). Therefore, the use of a multifaceted approach or multitargeted agents that affect specific tumor cell characteristics will likely be necessary to successfully eradicate GBM. The objective of this study was to investigate the usefulness of sulforaphane (SFN)-a constituent of cruciferous vegetables with a multitargeted effect-as a therapeutic agent for GBM. METHODS The inhibitory effects of SFN on established cell lines, early primary cultures, CD133-positive GSCs, GSC-derived spheroids, and GBM xenografts were evaluated using various methods, including GSC isolation and the sphere-forming assay, analysis of reactive oxygen species (ROS) and apoptosis, cell growth inhibition assay, comet assays for assessing SFN-triggered DNA damage, confocal microscopy, Western blot analysis, and the determination of in vivo efficacy as assessed in human GBM xenograft models. RESULTS SFN triggered the significant inhibition of cell survival and induced apoptotic cell death, which was associated with caspase 3 and caspase 7 activation. Moreover, SFN triggered the formation of mitochondrial ROS, and SFN-triggered cell death was ROS dependent. Comet assays revealed that SFN increased single- and double-strand DNA breaks in GBM. Compared with the vehicle control cells, a significantly higher amount of γ-H2AX foci correlated with an increase in DNA double-strand breaks in the SFN-treated samples. Furthermore, SFN robustly inhibited the growth of GBM cell-induced cell death in established cell cultures and early-passage primary cultures and, most importantly, was effective in eliminating GSCs, which play a major role in drug resistance and disease recurrence. In vivo studies revealed that SFN administration at 100 mg/kg for 5-day cycles repeated for 3 weeks significantly decreased the growth of ectopic xenografts that were established from the early passage of primary cultures of GBM10. CONCLUSIONS These results suggest that SFN is a potent anti-GBM agent that targets several apoptosis and cell survival pathways and further preclinical and clinical studies may prove that SFN alone or in combination with other therapies may be potentially useful for GBM therapy.

Abstract A29: Exploiting oxidative stress using MTH1 inhibitors in colorectal cancer

Abstract Background: Tumors are addicted to pathways neutralizing oxidative stress as a result of their high levels of reactive oxygen species (ROS). These ROS can oxidize nucleotides (NTs) within the precursor pool, which can cause cell death when incorporated into DNA. This tumor specific vulnerability might be exploited therapeutically. The mutT-homologue (MTH1) enzyme is recently identified as a non-oncogene target, which prevents incorporation of oxidized NTs into DNA (Gad et al, 2014; Huber et al, 2014). MTH1 inhibition by TH588 and (S)-Crizotinib caused tumor-specific oxidative DNA damage and cell death (Gad et al, 2014; Huber et al, 2014). Interestingly, hypoxia might enhance sensitivity to MTH1 inhibition (Qiu et al, 2015; Bräutigam et al, 2016). Thus, exploiting tumor oxidative stress via inhibition of MTH1 might be an exciting new approach to fight colorectal cancer (CRC) even in the presence of hypoxia. Aims and Hypotheses: In the present study we aim to compare the ability of TH588 HCl and (S)-Crizotinib to inhibit CRC growth in vitro and in vivo and to assess their mechanism(s) of action, including MTH1-dependent and -independent effects. We hypothesize that both compounds have the potency to inhibit CRC growth, conceivably via different mechanisms of action, which might not dependent on MTH1 inhibition (Kettle et al, 2016). Methods: All data were obtained using a human CRC liver metastasis-derived 3D spheroid line untreated or treated for 3 days with TH588 HCl or (S)-Crizotinib. Spheroids were pre-cultured and treated under normoxia (21% O2), hypoxia (0.1% O2) or only pre-cultured under hypoxia to mimic hypoxia and reoxygenation respectively. Results: Using a CellTiter-Glo cell viability assay we showed that increasing concentrations of (S)-Crizotinib under normoxia reduced cell viability with 23%, 64%, and 98% for 5, 10 and 20 μM respectively. By contrast, the maximal reduction of cell viability after treatment with TH588 HCl was only 32%. Interestingly, the dose response curve of (S)-Crizotinib was steeper when spheroids were reoxygenated just before treatment compared to normoxia and hypoxia, which implies that (S)-Crizotinib is more efficient. Importantly, hypoxia completely abolished the TH588-induced reduction in cell viability seen during normoxia. While the immediate effects on cell viability were different, both drugs (10 μM) severely reduced clone-forming efficiency both under normoxia and after hypoxia-reoxygenation prior to treatment. These results suggest different mechanisms of action of both drugs. As expected, both drugs caused an increase in DNA double-strand breaks, as measured by γH2AX staining. While chemotherapy treatment usually enriches the content of cancer stem cells, so far we did not observe this with either MTH1 inhibitor. Discussion and Conclusions: Until now, most research involving MTH1 inhibitors has been performed using cancer cell lines. In the present study we use patient-derived CRC spheroids and organoids. We previously found that organoids derived from patients with a poor-prognosis type of CRC are generally characterized by high expression of a H2O2 stress signature (Emmink et al, 2014). Exploiting this oxidative stress phenotype via inhibition of MTH1 might be an attractive new therapeutic avenue. Our results suggest that TH588 HCl and (S)-Crizotinib induce DNA damage and effectively reduce clone-forming potential of a CRC spheroid line, which was not limited by hypoxia pre-culturing. Interestingly, both compounds appear to act via different mechanisms. Further research should unravel these mechanisms of action. Citation Format: Lizet M. van der Waals, Danielle A.E. Raats, Jennifer M.J. Jongen, Tobias B. Dansen, Inne H.M. Borel Rinkes, Onno W. Kranenburg. Exploiting oxidative stress using MTH1 inhibitors in colorectal cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A29.

The Cytotoxic Effect of the PARP Inhibitor ABT-888 on Myeloproliferative Neoplasms

▪DNA instability, including increased DNA double strand breaks, has recently been demonstrated in myeloproliferative neoplasms (MPN). Because PARP1 has a central role in DNA repair and maintaining genomic integrity, we tested whether DNA repair inhibition with the PARP inhibitor ABT-888 (Abbvie, Chicago, IL) could affect the growth of myeloid neoplasms in-vitro and in-vivo, and/or enhance the cytotoxic effect of a standard alkylating agent commonly used in preparative regimens for stem cell transplants.Since the JAK2V617Fmutation is common in chronic MPNs or acute myeloid leukemia (AML) secondary to MPNs, we initially utilized JAK2 mutated AML cell lines (SET2 and HEL) in-vitro and in-vivo. We then tested the effect of PARP inhibition on CD34+ cells obtained from patients with primary myelofibrosis.JAK2 mutated SET2 and HEL cells were treated with ABT-888 in liquid cultures. Both cell lines were relatively sensitive with IC50 of 11.3μM and 74.2μM respectively. When the cells were treated with a combination of increasing doses of busulfan and 8μM ABT-888, significant synergy was observed, with busulfan IC50 decreasing from 27μM to 4μM in SET2 cells and from 45.1μM to 28.1μM in HEL cells. Treatment of SET2 cells with 8μM ABT-888, or with 8μM ABT888 and 10μM busulfan, was also accompanied by marked increase in γH2AX foci compared to control at 24 hours. We subsequently showed that ABT-888, busulfan and combination treatments resulted in increasing numbers of cells in G2/M arrest (30% vs. 35% vs 53% respectively, p=0.002). Western blot analysis of cells in G/2M arrest after combination treatment demonstrated a strongly increased phosphorylation of checkpoint kinase 1 (Chk1). In an immunofluorescence assay on treated cells we then showed that activated Chk1 translocated to the nucleus, suggesting that the Chk1-ATR pathway may be responsible for cell cycle arrest. To demonstrate the in-vivo effect of PARP inhibition, we transplanted 5x106 SET2 cells into a NOD/SCID/ gamma null mice to create a xenograft model. Mice developed AML with splenomegaly and bone marrow engraftment resulting in a median survival of 36 days. In mice that were treated with daily intraperitoneal injections of low dose ABT-888 (1.5mg/kg), starting 14 days after leukemic cell injection, survival increased to a median of 40 days (p=0.002).We subsequently tested the effect of PARP inhibition on primary myelofibrosis CD34+ cells obtained from the bone marrow or peripheral blood of 3 patients with JAK2V617Fmutation and 2 with CALR mutation. Control experiments were performed with normal CD34+ progenitors. In a standard clonogenic assay in methylcellulose, cells were plated with or without 4μM ABT-888, 5μM busulfan or a combination of both. Compared to untreated cells, ABT-888, busulfan and the combination of the two drugs reduced colony formation by 39%, 70% and 89%, respectively (p=0.01). Both CALR and JAK2 mutated cells were sensitive to ABT-888. On the contrary, treatment of normal CD34+ cells with ABT-888 did not affect colony growth.Here we describe the potential for PARP inhibition to exploit the genomic instability of MPNs. Potential clinical applications include the addition of PARP inhibition to busulfan in pretransplant conditioning or novel drug combinations including PARP inhibitors. DisclosuresNo relevant conflicts of interest to declare.

CXCR4/CXCL12 axis counteracts hematopoietic stem cell exhaustion through selective protection against oxidative stress.

Hematopoietic stem cells (HSCs) undergo self-renewal to maintain hematopoietic homeostasis for lifetime, which is regulated by the bone marrow (BM) microenvironment. The chemokine receptor CXCR4 and its ligand CXCL12 are critical factors supporting quiescence and BM retention of HSCs. Here, we report an unknown function of CXCR4/CXCL12 axis in the protection of HSCs against oxidative stress. Disruption of CXCR4 receptor in mice leads to increased endogenous production of reactive oxygen species (ROS), resulting in p38 MAPK activation, increased DNA double-strand breaks and apoptosis leading to marked reduction in HSC repopulating potential. Increased ROS levels are directly responsible for exhaustion of the HSC pool and are not linked to loss of quiescence of CXCR4-deficient HSCs. Furthermore, we report that CXCL12 has a direct rescue effect on oxidative stress-induced HSC damage at the mitochondrial level. These data highlight the importance of CXCR4/CXCL12 axis in the regulation of lifespan of HSCs by limiting ROS generation and genotoxic stress.