Induction Of γH2AX Research Articles

New mechanism of γ-H2AX generation: Surfactant-induced actin disruption causes deoxyribonuclease I translocation to the nucleus and forms DNA double-strand breaks

We previously showed that nonionic surfactants, nonylphenol polyethoxylates (NPEOs), induced phosphorylation of histone H2AX, forming γ-H2AX. In this study, we analyzed the mechanism of γ-H2AX generation by an NPEO with 15 ethylene oxide units (NPEO(15)). In MCF-7 breast carcinoma cells, NPEO(15) treatment induced γ-H2AX in a dose-dependent manner. EDTA and ZnCl2, two inhibitors of deoxyribonuclease I (DNase I), inhibited both the γ-H2AX and DNA double-strand breaks induced by NPEO(15). NPEO(15) disrupted filamentous actin and released free DNase I as detected by cell fractionation analysis. Based on immunofluorescence staining of DNase I and monitoring DNase I-GFP localization, DNase I was translocated from the cytosol to the nucleus of cells after treatment with NPEO(15). This translocation did not occur with the common DNA damage inducers ultraviolet B irradiation and hydrogen peroxide. Other surfactants, Tween 20, Triton X-100 and Nonidet P-40, also generated γ-H2AX. These results show that γ-H2AX induction by surfactants including NPEOs, occurs via a new mechanism involving release of free DNase I with actin disruption. This mechanism is distinct from the process of γ-H2AX generation caused by direct chemically induced DNA damage.

Evaluation of genotoxicity using automated detection of γH2AX in metabolically competent HepaRG cells.

The in situ detection of γH2AX was recently reported to be a promising biomarker of genotoxicity. In addition, the human HepaRG hepatoma cells appear to be relevant for investigating hepatic genotoxicity since they express most of drug metabolizing enzymes and a wild type p53. The aim of this study was to determine whether the automated in situ detection of γH2AX positive HepaRG cells could be relevant for evaluation of genotoxicity after single or long-term repeated in vitro exposure compared to micronucleus assay. Metabolically competent HepaRG cells were treated daily with environmental contaminants and genotoxicity was evaluated after 1, 7 and 14 days. Using these cells, we confirmed the genotoxicity of aflatoxin B1 and benzo(a)pyrene and demonstrated that dimethylbenzanthracene, fipronil and endosulfan previously found genotoxic with comet or micronucleus assays also induced γH2AX phosphorylation. Furthermore, we showed that fluoranthene and bisphenol A induced γH2AX while no effect had been previously reported in HepG2 cells. In addition, induction of γH2AX was observed with some compounds only after 7 days, highlighting the importance of studying long-term effects of low doses of contaminants. Together, our data demonstrate that automated γH2AX detection in metabolically competent HepaRG cells is a suitable high-through put genotoxicity screening assay.

Abstract 5437: A novel method to quantify gamma H2AX foci in circulating tumor cells in patients receiving chemotherapy for colorectal cancer

Abstract Phosphorylated H2AX (γH2AX) is a marker of DNA double-strand break damage and has been proposed as a pharmacodynamic biomarker following treatment with chemotherapy. γH2AX levels in circulating tumor cells (CTCs) may provide a sensitive direct marker for assessing drug effects in tumor tissue without the need for biopsy. In the present study, we established a novel assay to quantify changes in γH2AX in CTCs from patients with metastatic colorectal cancers undergoing treatment with Fulfox or Folfiri. γH2AX foci were initially assessed in human colon adenocarcinoma cancer cell lines (HT-29) treated with different concentrations of Oxaliplatin and SN-38 at different time points. Image capture was conducted using Confocal Microscope Leica SPE2 and γH2AX foci were analyzed with CellProfiler software. The highest number of γH2AX foci/nucleus was induced at 5uM for Oxaliplatin and 0.01uM for SN-38 and the peak of γH2AX foci/nucleus was observed between two-six hours post dose. To reproduce the analytical process for CTCs, we evaluated the γH2AX signal using both the CellSearch System (Janssen Diagnostics) and the DEPArray™ System (Silicon Biosystems). HT-29 cells were used, either as untreated controls or following 2 hours treatment with Oxaliplatin 5uM or SN-38 0.01uM and spiked in to healthy donor blood. Cells were defined as positive for γH2AX on the CellSearch according to a previously validated assay. For Oxaliplatin and SN-38 treated cells, 15.28% and 18.37% respectively were classified as positive for γH2AX, compared with 5.10% for untreated controls. However, using CellSearch, the fluorescent signal could not be quantified and we therefore repeated the experiment using the DEPArray platform. HT-29 cells were treated with SN-38 as above, and compared with an untreated control group using two different exposure times for fluorescein isothiocyanate-conjugated antibody (FITC): FITCI (100 ms and gain 1X) and FITCII (800 ms and gain 4X). The treated group showed a significantly increased intensity of FITC staining compared with the untreated control group: mean 363 vs mean 220 (p<0.0001) for FITCI, and mean 5521 vs mean 4365 (p<0.0040) for FITCII. The DEPArray system therefore has an advantage in being able to quantify differences in signal intensity caused by induction of γH2AX in CTCs and then select individual cells for further downstream analysis. This analysis is now being applied to patient samples collected pre and post-chemotherapy to evaluate the utility of the assay in a clinical setting. Citation Format: Matilde Saggese, Leah Ensell, Clare Vesely, Victoria Spanswick, Elena Peruzzi, Diana Cunati, Giulio Signorini, John Hartley, Hendrik Tobias Arkenau, Tim Meyer. A novel method to quantify gamma H2AX foci in circulating tumor cells in patients receiving chemotherapy for colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5437. doi:10.1158/1538-7445.AM2015-5437

γ-H2AX is a sensitive marker of DNA damage induced by metabolically activated 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a nicotine-derived nitrosamine, is a potent pulmonary carcinogen present in tobacco smoke. DNA adducts induced by metabolically activated NNK cause carcinogenesis; however, the DNA adducts are difficult to detect in cultured cells because of low intrinsic metabolic enzyme activity. In this study, we indirectly detected NNK-induced DNA adducts via the phosphorylation of histone H2AX (γ-H2AX) in A549 human lung adenocarcinoma epithelial cells. NNK treatment dose-dependently induced γ-H2AX. This γ-H2AX induction was suppressed by ataxia telangiectasia mutated inhibitors, suggesting that DNA double-strand breaks (DSBs) are formed during replication and repair of DNA adducts; however, DSBs could not be directly detected by biased sinusoidal field gel electrophoresis (BSFGE). CYP2A13-overexpressing cells showed prolonged γ-H2AX induction compared with control cells, and DSBs could be detected by BSFGE in CYP2A13-overexpressing cells as a clear migration of double-stranded DNA. These findings suggest that γ-H2AX is a sensitive marker of DNA adducts and provides a possible system for genotoxicity screening of chemicals such as NNK, which need metabolic activation to induce DNA damage.

Development of a multiparameter flow cytometric assay as a potential biomarker for homologous recombination deficiency in women with high-grade serous ovarian cancer.

ObjectivesPARP inhibitors (PARPi) are a novel class of drugs with activity in patients with acquired or germline homologous recombination (HR) deficiency-associated high-grade serous ovarian cancer (HGSOC). We hypothesized that measuring γH2AX as an indicator of DNA double-strand breaks (DSB), and MRE11 or RAD51 as an indicator of DSB repair, would reflect HR status and predict response to PARPi-based therapy. Our aim was to develop and use high-throughput multiparametric flow cytometry to quantify γH2AX with MRE11 or RAD51 in PBMCs as a readily available surrogate.MethodsHealthy donor PBMCs were used for assay development and optimization. We validated induction of γH2AX, MRE11 and RAD51 by staining with fluorophore-conjugated antibodies. The multiparameter flow cytometric method was applied to PBMC samples from recurrent HGSOC patients who were treated with PARPi, olaparib and carboplatin.ResultsStimulation was necessary for quantification of a DNA damage response to olaparib/carboplatin in healthy donor PBMCs. The flow cytometric protocol could not distinguish between cytoplasmic and nuclear RAD51, erroneously indicating activation in response to injury. Thus, MRE11 was selected as the marker of DSB repair. PBMCs from 15 recurrent HGSOC patients were then examined. Patients who did not respond to PARPi therapy had a significantly higher pre-treatment level of γH2AX (p = 0.01), and a higher ratio of γH2AX/MRE11 (11.0 [3.5–13.2] v. 3.3 [2.8–9.9], p < 0.03) compared with responders.ConclusionsWe successfully developed and applied a multiparameter flow cytometry assay to measure γH2AX and MRE11 in PBMCs. Prospective studies will be required to validate this surrogate biomarker assay as a potential predictive biomarker of PARPi-based therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0604-z) contains supplementary material, which is available to authorized users.

Hydroxyl metabolite of PCB 180 induces DNA damage signaling and enhances the DNA damaging effect of benzo[a]pyrene

Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) and their hydroxyl metabolites (OH-PCBs) are ubiquitous environmental contaminants in human tissues and blood. The toxicological impact of these metabolites is poorly understood. In this study rats were exposed to ultrapure PCB180 (10–1000mg/kgbw) for 28days and induction of genotoxic stress in liver was investigated. DNA damage signaling proteins (pChk1Ser317 and γH2AXSer319) were increased dose dependently in female rats. This increase was paralleled by increasing levels of the metabolite 3′-OH-PCB180. pChk1 was the most sensitive marker. In in vitro studies HepG2 cells were exposed to 1μM of PCB180 and 3′-OH-PCB180 or the positive control benzo[a]pyrene (BaP, 5μM). 3′-OH-PCB180, but not PCB180, induced CYP1A1 mRNA and γH2AX. CYP1A1 mRNA induction was seen at 1h, and γH2AX at 3h. The anti-oxidant N-Acetyl-l-Cysteine (NAC) completely prevented, and 17β-estradiol amplified the γH2AX induction by 3′-OH-PCB180. As 3′-OH-PCB180 induced CYP1A1, a major BaP-metabolizing and activating enzyme, interactions between 3′-OH-PCB180 and BaP was also studied. The metabolite amplified the DNA damage signaling response to BaP. In conclusion, metabolism of PCB180 to its hydroxyl metabolite and the subsequent induction of CYP1A1 seem important for DNA damage induced by PCB180 in vivo. Amplification of the response with estradiol may explain why DNA damage was only seen in female rats.

Non-canonical Bromodomain within DNA-PKcs Promotes DNA Damage Response and Radioresistance through Recognizing an IR-Induced Acetyl-Lysine on H2AX

Regulatory mechanisms underlying γH2AX induction and the associated cell fate decision during DNA damage response (DDR) remain obscure. Here, we discover a bromodomain (BRD)-like module in DNA-PKcs (DNA-PKcs-BRD) that specifically recognizes H2AX acetyl-lysine 5 (K5ac) for sequential induction of γH2AX and concurrent cell fate decision(s). First, top-down mass spectrometry of radiation-phenotypic, full-length H2AX revealed a radiation-inducible, K5ac-dependent induction of γH2AX. Combined approaches of sequence-structure modeling/docking, site-directed mutagenesis, and biochemical experiments illustrated that through docking on H2AX K5ac, this non-canonical BRD determines not only the H2AX-targeting activity of DNA-PKcs but also the over-activation of DNA-PKcs in radioresistant tumor cells, whereas a Kac antagonist, JQ1, was able to bind to DNA-PKcs-BRD, leading to re-sensitization of tumor cells to radiation. This study elucidates the mechanism underlying the H2AX-dependent regulation of DNA-PKcs in ionizing radiation-induced, differential DDR, and derives an unconventional, non-catalytic domain target in DNA-PKs for overcoming resistance during cancer radiotherapy.

Using gamma-H2AX and H2AX quantitative ELISA for monitoring DNA damage induced by chemotherapeutic agents and irradiation exposure.

2559 Background: Gamma-H2AX (γH2AX) is a biomarker for DNA double-strand breaks and programmed cell death, but variable relative amounts of H2AX in different samples causes ambiguity in the meaning of the γH2AX level unless it is related to total H2AX levels. We developed a 96-well plate-based ELISA for quantifying γH2AX and H2AX levels in crude extracts of tumor cells, CTCs and biopsy tissues and are validating it for applications in irradiation exposure monitoring and in pharmacodynamic evaluation of anti-cancer agents. Methods: The ELISA was used to analyze extracts of several NCI60 tumor cell lines that had been exposed to a variety of agents, including ionizing radiation, inhibitors of Top-1 (CPT, SN-38, Topotecan), PARP (ABT-888, AZD-2281, MK-4827) and ATR (VE-821, VE-822, AZD-6738, Compound 45, NU-6027), and their combinations. Combination regimens of CPT-11 with PARP inhibitors (ABT-888, AZD-2281, MK-4827) were further evaluated in vivo in the A375 xenograft mouse model. Patient samples obtained for research purposes were also examined by ELISA for feasibility and utility. Results: In vitro, dose-dependent increases in the ratio of γH2AX to H2AX were detected after escalating ionizing radiation exposure and concentration-dependent increases after Top-1 exposure. Treating with inhibitors of PARP or ATR alone did not significantly induce γH2AX. Combinations of Top-1 inhibitors with PARP or ATR inhibitors led to synergistic induction of DNA damage. Among five ATR inhibitors evaluated in combination with Top-1 inhibitors, VE-822 and AZD-6738 were observed to have the highest synergy for γH2AX induction, while NU-6027 showed none. Combinations of CPT-11 with ABT-888, AZD-2281 or MK-4827 showed synergistic induction of γH2AX in A375 xenografts in vivo. Additional testing of human specimens including PBMCs, bone marrow and tumor biopsies proved the assay’s clinical suitability and potential advantages. Conclusions: A newly developed quantitative ELISA for measuring both γH2AX and H2AX is ready for clinical validation for monitoring DNA damage induced by chemotherapeutic agents or irradiation exposure. Funded by NCI Contract No. HHSN261200800001E.

DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

Calibration of the γ-H2AX DNA double strand break focus assay for internal radiation exposure of blood lymphocytes.

DNA double strand break (DSB) formation induced by ionizing radiation exposure is indicated by the DSB biomarkers γ-H2AX and 53BP1. Knowledge about DSB foci formation in-vitro after internal irradiation of whole blood samples with radionuclides in solution will help us to gain detailed insights about dose-response relationships in patients after molecular radiotherapy (MRT). Therefore, we studied the induction of radiation-induced co-localizing γ-H2AX and 53BP1 foci as surrogate markers for DSBs in-vitro, and correlated the obtained foci per cell values with the in-vitro absorbed doses to the blood for the two most frequently used radionuclides in MRT (I-131 and Lu-177). This approach led to an in-vitro calibration curve. Overall, 55 blood samples of three healthy volunteers were analyzed. For each experiment several vials containing a mixture of whole blood and radioactive solutions with different concentrations of isotonic NaCl-diluted radionuclides with known activities were prepared. Leukocytes were recovered by density centrifugation after incubation and constant blending for 1 h at 37°C. After ethanol fixation they were subjected to two-color immunofluorescence staining and the average frequencies of the co-localizing γ-H2AX and 53BP1 foci/nucleus were determined using a fluorescence microscope equipped with a red/green double band pass filter. The exact activity was determined in parallel in each blood sample by calibrated germanium detector measurements. The absorbed dose rates to the blood per nuclear disintegrations occurring in 1 ml of blood were calculated for both isotopes by a Monte Carlo simulation. The measured blood doses in our samples ranged from 6 to 95 mGy. A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied. There were only minor nuclide-specific intra- and inter-subject deviations.

The nuclear oncogene SET controls DNA repair by KAP1 and HP1 retention to chromatin.

Cells experience damage from exogenous and endogenous sources that endanger genome stability. Several cellular pathways have evolved to detect DNA damage and mediate its repair. Although many proteins have been implicated in these processes, only recent studies have revealed how they operate in the context of high-ordered chromatin structure. Here, we identify the nuclear oncogene SET (I2PP2A) as a modulator of DNA damage response (DDR) and repair in chromatin surrounding double-strand breaks (DSBs). We demonstrate that depletion of SET increases DDR and survival in the presence of radiomimetic drugs, while overexpression of SET impairs DDR and homologous recombination (HR)-mediated DNA repair. SET interacts with the Kruppel-associated box (KRAB)-associated co-repressor KAP1, and its overexpression results inthe sustained retention of KAP1 and Heterochromatin protein 1 (HP1) on chromatin. Our results are consistent with a model in which SET-mediated chromatin compaction triggers an inhibition of DNA end resection and HR.

High Throughput Measurement of γH2AX DSB Repair Kinetics in a Healthy Human Population

The Columbia University RABiT (Rapid Automated Biodosimetry Tool) quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB) by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY) (r=0.257, P=0.02) and a negative correlation with residuals (r=-0.521, P=<0.0001). A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001). Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay.

Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation.

In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (γH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis.

Detection of benz[j]aceanthrylene in urban air and evaluation of its genotoxic potential.

Benz[j]aceanthrylene (B[j]A) is a cyclopenta-fused polycyclic aromatic hydrocarbon with strong mutagenic and carcinogenic effects. We have identified B[j]A in air particulate matter (PM) in samples collected in Stockholm, Sweden and in Limeira, Brazil using LC-GC/MS analysis. Determined concentrations ranged between 1.57 and 12.7 and 19.6-30.2 pg/m(3) in Stockholm and Limeira, respectively, which was 11-30 times less than benzo[a]pyrene (B[a]P) concentrations. Activation of the DNA damage response was evaluated after exposure to B[j]A in HepG2 cells in comparison to B[a]P. We found that significantly lower concentrations of B[j]A were needed for an effect on cell viability compared to B[a]P, and equimolar exposure resulted in significant more DNA damage with B[j]A. Additionally, levels of γH2AX, pChk1, p53, pp53, and p21 proteins were higher in response to B[j]A than B[a]P. On the basis of dose response induction of pChk1 and γH2AX, B[j]A potency was 12.5- and 33.3-fold higher than B[a]P, respectively. Although B[j]A levels in air were low, including B[j]A in the estimation of excess lifetime cancer risk increased the risk up to 2-fold depending on which potency factor for B[j]A was applied. Together, our results show that B[j]A could be an important contributor to the cancer risk of air PM.

A synthetic lethal screen reveals enhanced sensitivity to ATR inhibitor treatment in mantle cell lymphoma with ATM loss-of-function.

Mechanisms to maintain genomic integrity are essential for cells to remain viable. Not surprisingly, disruption of key DNA damage response pathway factors, such as ataxia telangiectasia-mutated (ATM)/ataxia telangiectasia and RAD3-related (ATR) results in loss of genomic integrity. Here, a synthetic lethal siRNA-screening approach not only confirmed ATM but identified additional replication checkpoint proteins, when ablated, enhanced ATR inhibitor (ATRi) response in a high-content γ-H2AX assay. Cancers with inactivating ATM mutations exhibit impaired DNA double-stranded break (DSB) repair and rely on compensatory repair pathways for survival. Therefore, impairing ATR activity may selectively sensitize cancer cells to killing. ATR inhibition in an ATM-deficient context results in phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) and leads to induction of γ-H2AX. Using both in vitro and in vivo models, ATR inhibition enhanced efficacy in ATM loss-of-function mantle cell lymphoma (MCL) compared with ATM wild-type cancer cells. In summary, single-agent ATR inhibitors have therapeutic utility in the treatment of cancers, like MCL, in which ATM function has been lost. These data suggest that single-agent ATR inhibitors have therapeutic utility and that ATR uses a complex and coordinated set of proteins to maintain genomic stability that could be further exploited.

A Novel Alkylating Agent Melphalan Flufenamide Ethyl Ester Induces an Irreversible DNA Damage in Multiple Myeloma Cells

Background and Rationale The alkylating agent melphalan is actively used in multiple myeloma therapy; however, dose-limiting toxicities and development of resistance limits its use. Melphalan flufenamide ethyl ester (melflufen) is an enzyme-activated analogue of melphalan which allows for a more rapid and higher intracellular accumulation of melphalan in tumor cells than is achievable by direct exposure to equimolar doses of melphalan. Our earlier study showed that melflufen is a more potent anti-MM agent than melphalan, that can overcome conventional drug resistance and induce synergistic anti-MM activity in combination with bortezomib, lenalidomide, or dexamethasone (Chauhan et al, Clinical Cancer Res 2013, 19(11): 3019-3031). These preclinical studies provided the basis for an ongoing phase 1 clinical trial of melflufen in MM. Here we examined whether the potent anti-MM activity of melflufen versus melphalan is due to their differential effect on DNA damage and repair signaling pathways.Material and Methods We utilized melphalan-sensitive (MM.1S, RPMI-8226) and melphalan-resistant (LR-5) human MM cell lines. Immunoblot analysis was performed using antibodies specific against γ-H2AX, ATR, CHK1, Ku80, or GAPDH. Cell viability, cell cycle analysis were performed using MTT and propidium iodide staining. Cell viability, cell cycle and immunoblot studies were performed using equimolar concentrations of melflufen to melphalan, as in our prior study (Chauhan et al, Clinical Cancer Res 2013, 19(11): 3019-3031). Statistical significance was determined from Student’s t test. Melflufen was obtained from Oncopeptides AB, and melphalan was purchased from Sigma Chemical Company.Results Melflufen triggered cytotoxicity in MM cell lines including, melphalan-resistant LR-5 cells. An early event in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX (γ-H2AX) at the sites in proximity to DNA breaks. Melflufen-induced apoptosis was associated with dose- and time- dependent rapid phosphorylation of γ-H2AX. We next compared the kinetics of induction of γ-H2AX/ATR/CHK1/Ku80 in melflufen- versus melphalan-treated MM cells. Cells were treated with equimolar concentrations of melflufen or melphalan for 2h, 4h, 6h, and 24h; protein lysates were prepared and subjected to immunoblot analysis with antibodies specific against γ-H2AX, ATR, CHK1, Ku80, GAPDH. Melflufen, but not melphalan, triggered induction of γ-H2AX, ATR, and CHK1 as early as after 2h exposure in both melphalan-sensitive and –resistant cells. Melphalan triggered induction of γ-H2AX in MM.1S and RPMI-8826 cells at 6h and 24h, respectively; and as expected, no induction of γ-H2AX was observed in LR-5 melphalan-resistant cells even after 24h treatment. Similar kinetics were observed for ATR and CHK1. We next examined the induction of DNA repair pathway in response to melflufen and melphalan using Ku80 as a marker protein. Melphalan, but not melflufen, triggered induction of Ku80. These data suggest that 1) melflufen triggers an early, and rapid DNA damage versus melphalan; 2) while melphalan induces DNA repair, no mechanism of DNA repair process is activated in response to melflufen treatment. Reports that the mechanisms for melphalan-resistance includes activation of DNA repair pathways, coupled with our findings showing lack of DNA repair pathway in melflufen-treated cells, suggest that melflufen overcomes melphalan-resistance, at least in part, by triggering an irreversible DNA damage. To further validate these data, we performed drug washout experiments and analysis of cell viability. Cells were treated with equimolar doses of melflufen or melphalan for 1h; cells were then washed with plain medium to remove drugs, cultured in fresh medium for additional 48h, and then analyzed for viability. One hour treatment of cells with melflufen, but not melphalan, triggers cytotoxicity in MM cells. Similar findings were observed using cell cycle analysis: % Sub G0/G1 after melflufen treatment was 81.1% in MM.1S; 80% in RPMI-8226; and 77% in LR-5 cells. Of note, melphalan-treated cells triggered only minimal (10%) accumulation of cells in Sub-G0/G1 phase.Conclusion Our data therefore suggest that melflufen triggers a rapid, robust and an irreversible DNA damage, which may account for its ability to overcome melphalan-resistance in MM cells. DisclosuresNordström:Oncopeptides AB: Employment. Lindberg:Oncopeptides AB: Employment. Chauhan:Oncopeptides AB: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

ATR Inhibition Exacerbates Replication Stress in TP53 or ATM Deficient CLL Cells and Enhances Sensitivity to Chemotherapy and Targeted Therapy

DNA damage response (DDR) defects, particularly TP53 or biallelic ATM aberrations, are associated with chemoresistance in chronic lymphocytic leukemia (CLL). Chemoimmunotherapy or B-cell receptor signaling inhibitors alone may not be sufficient to overcome adverse prognosis or provide durable response in TP53 or biallelic ATM inactivated CLL. In particular, genomic instability resulting from impaired DDR facilitates rapid clonal evolution leading to treatment refractoriness or disease relapse. Development of therapeutic approaches specifically targeting DDR defects is therefore necessary for effective long-term control of DDR-defective CLL.We previously demonstrated selective cytotoxicity of the ATR inhibitor AZD6738 towards TP53 or ATM null CLL cells, and validated this in CLL xenograft models for biallelic TP53 or ATM loss. Here, we provide mechanistic insight into the synthetically lethal interactions between ATR pathway inhibition and TP53 or ATM loss in CLL, and offer experimental evidence supporting the use of ATR inhibition in combination with conventional chemotherapies and other targeted therapies in CLL.To determine the mechanism of ATR inhibition in CLL, we first investigated its effect on DDR-intact cycling primary cells. We observed compensatory activation of the ATM/p53 pathway in AZD6738-treated cells in response to hydroxyurea, providing evidence for crosstalk between ATR and ATM/p53 pathways. Next, we explored the cellular consequence of ATR inhibition in CLL cells with DDR loss. To determine the impact of ATR inhibition on DNA replication, we performed DNA fiber analysis, which revealed significantly increased replication fork stalling and firing of replication origins upon AZD6738 treatment in ATM/p53-defective CLL. To investigate the effect of ATR inhibition on DNA damage, we measured γH2AX and 53BP1 foci formation, markers of DNA double-strand breaks. Significant induction of γH2AX and 53BP1 foci was seen in ATM/p53-defective CLL cells upon AZD6738 treatment, suggesting that ATR inhibition exacerbates replication stress in ATM/p53 defective cells by imposing requirement for DDR through the ATM/p53 pathway.On the contrary, 53BP1 bodies, a marker of unreplicated DNA, appeared in ATM/p53 proficient but not in ATM/p53 deficient cells following AZD6738 exposure, indicating that cell cycle arrest in response to replication stress is ATM/p53 dependent. This was corroborated by cell cycle profiling and co-labeling experiments of γH2AX with phosphohistone H3 ser10, a marker of mitosis, showing that ATM/p53-defective CLL cells carrying unrepaired DNA damage continued to cycle. Thus, ATR inhibition results in accumulation of intolerable levels of DNA damage in DDR-defective CLL, leading to cell death by mitotic catastrophe, which we have confirmed both in vitro and in AZD6738-treated murine xenograft models.The benefit of ATR inhibition as a therapeutic strategy for DDR-defective CLL lies in its ability to alter the subclonal landscape in favor of less unstable DDR-proficient subclones, which are less susceptible to clonal evolution, thus reducing the likelihood of disease relapse. We reasoned that this provides a strong rationale for addition of AZD6738 to existing therapeutic agents for the treatment of CLL with DDR defects. We demonstrated synergistic and additive effects of low-dose AZD6738 in ATM/p53-defective CLL cells with DNA damaging agents such as chlorambucil, fludarabine, bendamustine and cyclophosphamide, BCR-signaling inhibitors and the PARP inhibitor olaparib. We validated the AZD6738 plus chlorambucil combination in biallelic TP53 or ATM inactivated primary CLL xenografts, where combined AZD6738/chlorambucil treatment was superior to chlorambucil alone, as evidenced by significantly greater reduction in tumor load and percentage of CLL subclones with del(17p) or del(11q) in animals treated with combination regimen compared to single-agent chlorambucil. Similar investigations evaluating AZD6738 plus ibrutinib versus ibrutinib monotherapy in primary CLL xenografts are currently underway.We conclude that ATR inhibition is a suitable approach for targeting the loss of p53 function in aggressive CLL subclones and should be considered as a valuable addition to DNA damaging agents and current targeted treatments. DisclosuresOff Label Use: ATR inhibitor AZD6738 targets TP53-null or ATM-null phenotype inducing synthetic lethality. Brown:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.

NNK-Induced DNA Methyltransferase 1 in Lung Tumorigenesis in A/J Mice and Inhibitory Effects of (−)-Epigallocatechin-3-Gallate

DNA methyltransferase 1 (DNMT1), a key enzyme mediating DNA methylation, is known to be elevated in various cancers, including the mouse lung tumors induced by the tobacco-specific carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). However, it is not known whether DNMT1 expression is induced right after NNK treatment and how DNMT1 expression varies throughout lung tumorigenesis. In the present study, we found that administration of NNK to A/J mice caused elevation of DNMT1 in bronchial epithelial cells at Days 1, 3, and 14 after NNK treatment. DNMT1 elevation at Day 1 was accompanied by an increase in phospho-histone H2AX (γ-H2AX) and phospho-AKT (p-AKT). At Weeks 5 to 20, NNK-induced DNMT1 in lung tissues was in lower levels than the early stages, but was highly elevated in lung tumors at Week 20. In addition, the early induction of p-AKT and γ-H2AX as well as cleaved caspase-3 in NNK-treated lung tissues was not detected at Weeks 5 to 20 but was elevated in lung tumors. In concordance with DNMT1 elevation, promoter hypermethylation of tumor suppressor genes Cdh13, Prdm2, and Runx3 was observed in lung tissues at Day 3 and in lung tumors. Treatment by EGCG attenuated DNMT1, p-AKT, and γ-H2AX inductions at Days 1 and 3 and inhibited lung tumorigenesis.

In vivo evidence that DNA polymerase kappa is responsible for error-free bypass across DNA cross-links induced by mitomycin C.

Translesion DNA synthesis (TLS) is an important pathway that avoids genotoxicity induced by endogenous and exogenous agents. DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in TLS but its protective roles against DNA damage in vivo are still unclear. To better understand these roles, we have established knock-in mice that express catalytically-inactive Polk and crossbred them with gpt delta mice, which possess reporter genes for mutations. The resulting mice (inactivated Polk KI mice) were exposed to mitomycin C (MMC), and the frequency of point mutations, micronucleus formation in peripheral erythrocytes, and γH2AX induction in the bone marrow was determined. The inactivated Polk KI mice exhibited significantly higher frequency of mutations at CpG and GpG sites, micronucleated cells, and γH2AX foci-positive cells than did the Polk wild-type (Polk(+)) mice. Recovery from MMC-induced DNA damage, which was evaluated by γH2AX induction, was retarded in embryonic fibroblasts from the knock-in mice when compared to those from the Polk(+) mice. These results suggest that Polk mediates TLS, which suppresses point mutations and DNA double-strand breaks caused by intra- and interstrand cross-links induced by MMC treatment. The established knock-in mice are extremely useful to elucidate the in vivo roles of the catalytic activity of Polk in suppressing DNA damage that was induced by a variety of genotoxic stresses.

Abstract 4689: Pre-clinical investigation of the wee1 inhibitor MK-1775 using pharmacodynamic and mechanistic markers in diverse cancer models in vivo

Abstract Wee1 kinase is a critical regulator of the G2/M checkpoint by initiating inhibitory phosphorylation of the conserved tyrosine-15 residue of cdk1/cdc2. Wee1 also phosphorylates tyrosine 15 of cdk2 and plays a crucial role in maintaining genome integrity during S-phase. MK1775 (NSC 754352) is a small molecule selective inhibitor of Wee1 kinase that is currently under clinical evaluation. We have developed a quantitative immunofluorescence assay to measure inhibition of Wee1 by detection of pY15 of cyclin-dependent kinases. A full analysis of the pharmacodynamic (PD) effects of Wee1 inhibition by MK1775, as well as the downstream mechanistic consequences of Wee1 inhibition on DNA repair, apoptosis, and premature mitotic entry markers was performed using two xenograft models: A673 Ewing sarcoma and U87-MG glioblastoma. Four DNA repair markers (pNbs1, γH2AX, Rad51, and pATR) were examined as well as the mitotic marker pHistone H3. Advanced quantitative image analysis was performed using Definiens software to measure changes in PD markers by two approaches: total nuclear area measurements and foci per nucleus. Definiens software enables enhanced analysis of the markers by enumeration of the nuclei in the imaged xenograft tissues over an entire data set with high-content capacity. Our data demonstrate greater than 80% inhibition of pY15-cdk in vivo at the clinically relevant dose of 60 mg/kg (180 mg/m2) MK1775 in two xenograft models. In addition, γH2AX induction was observed after multiple doses of MK1775 as a single agent. A PD biomarker time course was determined for MK1775 and gemcitabine (NSC 750927) as single agents to develop a drug administration schedule for combination studies in a Ewing sarcoma model. The time point at which the DNA damage response peaked following administration of the maximum tolerated dose of gemcitabine in the mouse was determined. This study elucidates a broad profile of PD marker response, as well as the corresponding levels of MK1775 in the xenografts. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Deborah F. Wilsker, Allison M. Marrero, Melinda Hollingshead, Scott M. Lawrence, Alice Chen, Shivaani Kummar, Joseph M. Covey, Ralph E. Parchment, Robert J. Kinders, Joseph E. Tomaszewski, James H. Doroshow. Pre-clinical investigation of the wee1 inhibitor MK-1775 using pharmacodynamic and mechanistic markers in diverse cancer models in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4689. doi:10.1158/1538-7445.AM2014-4689