Cellular DNA Damage Response Pathways Research Articles

Context matters: DNA virus infection reshapes DNA damage response pathways

The cellular DNA damage response pathway can have vastly different outcomes depending on the source of its activation. Justice and colleagues apply phosphoproteomics to uncover a divergence in DNA-PK and ATM kinase activities in the contexts of DNA damage and DNA virus infection.

A mathematical model to investigate the effects of ceralasertib and olaparib in targeting the cellular DNA Damage responses pathway.

The ataxia-telangiectasia and Rad3-related (ATR) inhibitor ceralasertib and the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib have shown synergistic activity, in vitro, in the FaDu ATM-KO cell line. It was found that combining these drugs with lower doses and for shorter treatment periods induced greater or equal toxicity in cancer cells than using either as a single agent. Here, we develop a biologically-motivated mathematical model governed by a set of ordinary differential equations, considering the cell cycle-specific interactions of olaparib and ceralasertib. By exploring a range of different possible drug mechanisms, we have studied the effects of their combination as well as which drug interactions are the most prominent. After careful model selection, the model was calibrated and compared to relevant experimental data. We have used this developed model further to investigate other doses of olaparib and ceralasertib in combination, which can be potentially helpful in exploring optimised dosage and delivery. Significance Statement Drugs that target cellular DNA damage repair pathways are now being used as new ways to maximise the effect of multimodality treatments such as radiotherapy. Here, we develop a mathematical model to investigate the effects of ceralasertib and olaparib, two drugs that target DNA damage response pathways.

Evidence for a Hepatitis B Virus Short RNA Fragment Directly Targeting the Cellular RRM2 Gene.

The hepatitis B virus (HBV) is one of the smallest but most highly infectious human pathogens. With a DNA genome of only 3.2 kb and only four genes, HBV successfully completes its life cycle by using intricate processes to hijack the host machinery. HBV infects non-dividing liver cells in which dNTPs are limited. As a DNA virus, HBV requires dNTPs for its replication. HBV induces the ATR-mediated cellular DNA damage response pathway to overcome this constraint. This pathway upregulates R2 (RRM2) expression in generating an active RNR holoenzyme catalyzing de novo dNTP synthesis. Previously we reported that ERE, a small RNA fragment within the HBx ORF, is sufficient to induce R2 upregulation. Interestingly, there is high sequence similarity between ERE and a region within the R2 5′UTR that we named R2-box. Here, we established a mutant cell line in the R2-box region of the R2 gene using CRISPR-Cas9 technology to investigate the R2 regulation by ERE. This cell line expresses a much lower R2 level than the parental cell line. Interestingly, the HBV infection and life cycle were severely impaired. These cells became permissive to HBV infection upon ectopically R2 expression. These results validate the requirement of the R2 gene expression for HBV replication. Remarkably, the R2-box mutated cells became ERE refractory, suggesting that the homology region between ERE and R2 gene is critical for ERE-mediated R2 upregulation. Thus, along with the induction of the ATR pathway of the DNA damage response, ERE might also directly target the R2 gene via the R2-box.

PARP1 Upregulation in Recurrent Oral Cancer and Treatment Resistance.

First-line treatments for oral cancer typically include surgery, radiation, and in some cases, chemotherapy. Radiation and oral cancer chemotherapeutics confer cytotoxicity largely by inducing DNA damage, underscoring the importance of the cellular DNA damage repair and response pathways in cancer therapy. However, tumor recurrence and acquired resistance, following the initial response to treatment, remains as a major clinical challenge. By analyzing oral tumor cells derived from the primary and recurrent tumors of the same patient, our study revealed upregulated PARP1 expression in the recurrent tumor cells. Cisplatin and 5-fluorouracil treatment further augmented PARP1 expression in the recurrent, but not the primary, tumor cells. Post-treatment upregulation of PARP1 was dependent on the catalytic activities of PARP and CDK7. Consistent with the established function of PARP1 in DNA repair, we showed that overexpression of PARP1 rendered the primary tumor cells highly resistant to DNA damage treatment. Conversely, PARP inhibition partially reversed the treatment resistance in the recurrent tumor cells; combinatorial treatment using a PARP inhibitor and cisplatin/5-fluorouracil significantly sensitized the tumor response in vivo. Taken together, we reported here PARP1 upregulation as a clinically relevant mechanism involved in oral cancer recurrence, and suggested the clinical benefit of PARP inhibitors, currently approved for the treatment of several other types of cancer, in oral cancer.

The Promotion of Genomic Instability in Human Fibroblasts by Adenovirus 12 Early Region 1B 55K Protein in the Absence of Viral Infection.

The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K+HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K+HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K+HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K+HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K+HSFs. After DNA damage, appreciably more foci were formed in 55K+HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K+HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein–protein interactions, causing genomic instability.

CAG RNAs induce DNA damage and apoptosis by silencing NUDT16 expression in polyglutamine degeneration

DNA damage plays a central role in the cellular pathogenesis of polyglutamine (polyQ) diseases, including Huntington's disease (HD). In this study, we showed that the expression of untranslatable expanded CAG RNA per se induced the cellular DNA damage response pathway. By means of RNA sequencing (RNA-seq), we found that expression of the Nudix hydrolase 16 (NUDT16) gene was down-regulated in mutant CAG RNA-expressing cells. The loss of NUDT16 function results in a misincorporation of damaging nucleotides into DNAs and leads to DNA damage. We showed that small CAG (sCAG) RNAs, species generated from expanded CAG transcripts, hybridize with CUG-containing NUDT16 mRNA and form a CAG-CUG RNA heteroduplex, resulting in gene silencing of NUDT16 and leading to the DNA damage and cellular apoptosis. These results were further validated using expanded CAG RNA-expressing mouse primary neurons and in vivo R6/2 HD transgenic mice. Moreover, we identified a bisamidinium compound, DB213, that interacts specifically with the major groove of the CAG RNA homoduplex and disfavors the CAG-CUG heteroduplex formation. This action subsequently mitigated RNA-induced silencing complex (RISC)-dependent NUDT16 silencing in both in vitro cell and in vivo mouse disease models. After DB213 treatment, DNA damage, apoptosis, and locomotor defects were rescued in HD mice. This work establishes NUDT16 deficiency by CAG repeat RNAs as a pathogenic mechanism of polyQ diseases and as a potential therapeutic direction for HD and other polyQ diseases.

Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication.

Here, we show that the cellular DNA replication protein and ATR substrate SMARCAL1 is recruited to viral replication centers early during adenovirus infection and is then targeted in an E1B-55K/E4orf6- and cullin RING ligase-dependent manner for proteasomal degradation. In this regard, we have determined that SMARCAL1 is phosphorylated at S123, S129, and S173 early during infection in an ATR- and CDK-dependent manner, and that pharmacological inhibition of ATR and CDK activities attenuates SMARCAL1 degradation. SMARCAL1 recruitment to viral replication centers was shown to be largely dependent upon SMARCAL1 association with the RPA complex, while Ad-induced SMARCAL1 phosphorylation also contributed to SMARCAL1 recruitment to viral replication centers, albeit to a limited extent. SMARCAL1 was found associated with E1B-55K in adenovirus E1-transformed cells. Consistent with its ability to target SMARCAL1, we determined that E1B-55K modulates cellular DNA replication. As such, E1B-55K expression initially enhances cellular DNA replication fork speed but ultimately leads to increased replication fork stalling and the attenuation of cellular DNA replication. Therefore, we propose that adenovirus targets SMARCAL1 for degradation during infection to inhibit cellular DNA replication and promote viral replication.IMPORTANCE Viruses have evolved to inhibit cellular DNA damage response pathways that possess antiviral activities and utilize DNA damage response pathways that possess proviral activities. Adenovirus has evolved, primarily, to inhibit DNA damage response pathways by engaging with the ubiquitin-proteasome system and promoting the degradation of key cellular proteins. Adenovirus differentially regulates ATR DNA damage response signaling pathways during infection. The cellular adenovirus E1B-55K binding protein E1B-AP5 participates in ATR signaling pathways activated during infection, while adenovirus 12 E4orf6 negates Chk1 activation by promoting the proteasome-dependent degradation of the ATR activator TOPBP1. The studies detailed here indicate that adenovirus utilizes ATR kinase and CDKs during infection to promote the degradation of SMARCAL1 to attenuate normal cellular DNA replication. These studies further our understanding of the relationship between adenovirus and DNA damage and cell cycle signaling pathways during infection and establish new roles for E1B-55K in the modulation of cellular DNA replication.

Interaction of the Human Papillomavirus E1 Helicase with UAF1-USP1 Promotes Unidirectional Theta Replication of Viral Genomes.

Human papillomaviruses (HPVs) are important pathogens with a significant medical burden. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism. In this report, we provide evidence that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 deubiquitinating enzyme complex, a member of the Fanconi anemia DNA damage response pathway, is required for the completion of the bidirectional theta replication of the HPV11 genome and the subsequent initiation of the unidirectional replication. We show that unidirectional replication proceeds via theta structures and is supported by the cellular Bloom helicase, which interacts directly with E1 and whose engagement in HPV11 replication requires UAF1-USP1 activity. We propose that the unidirectional replication of the HPV11 genome initiates from replication fork restart events. These findings suggest a new role for the Fanconi anemia pathway in HPV replication.IMPORTANCE Human papillomaviruses (HPVs) are important pathogens that replicate their double-stranded circular DNA genome in the nucleus of infected cells. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism, and the onset of viral replication requires the engagement of cellular DNA damage response pathways. In this study, we showed that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 complex is necessary for the completion of bidirectional replication and the subsequent initiation of the unidirectional replication mechanism. Our results suggest HPVs may use the cellular Fanconi anemia DNA damage pathway to achieve the separation of daughter molecules generated by bidirectional theta replication. Additionally, our results indicate that the unidirectional replication of the HPV genome is initiated from restarted bidirectional theta replication forks.

The ATM and Rad3-Related (ATR) Protein Kinase Pathway Is Activated by Herpes Simplex Virus 1 and Required for Efficient Viral Replication.

The ATM and Rad3-related (ATR) protein kinase and its downstream effector Chk1 are key sensors and organizers of the DNA damage response (DDR) to a variety of insults. Previous studies of herpes simplex virus 1 (HSV-1) showed no evidence for activation of the ATR pathway. Here we demonstrate that both Chk1 and ATR were phosphorylated by 3 h postinfection (h.p.i.). Activation of ATR and Chk1 was observed using 4 different HSV-1 strains in multiple cell types, while a specific ATR inhibitor blocked activation. Mechanistic studies point to early viral gene expression as a key trigger for ATR activation. Both pATR and pChk1 localized to the nucleus within viral replication centers, or associated with their periphery, by 3 h.p.i. Significant levels of pATR and pChk1 were also detected in the cytoplasm, where they colocalized with ICP4 and ICP0. Proximity ligation assays confirmed that pATR and pChk1 were closely and specifically associated with ICP4 and ICP0 in both the nucleus and cytoplasm by 3 h.p.i., but not with ICP8 or ICP27, presumably in a multiprotein complex. Chemically distinct ATR and Chk1 inhibitors blocked HSV-1 replication and infectious virion production, while inhibitors of ATM, Chk2, and DNA-dependent protein kinase (DNA-PK) did not. Together our data show that HSV-1 activates the ATR pathway at early stages of infection and that ATR and Chk1 kinase activities play important roles in HSV-1 replication fitness. These findings indicate that the ATR pathway may provide insight for therapeutic approaches.IMPORTANCE Viruses have evolved complex associations with cellular DNA damage response (DDR) pathways, which sense troublesome DNA structures formed during infection. The first evidence for activation of the ATR pathway by HSV-1 is presented. ATR is activated, and its downstream target Chk1 is robustly phosphorylated, during early stages of infection. Both activated proteins are found in the nucleus associated with viral replication compartments and in the cytoplasm associated with viral proteins. We also demonstrate that both ATR and Chk1 kinase activities are important for viral replication. The findings suggest that HSV-1 activates ATR and Chk1 during early stages of infection and utilizes the enzymes to promote its own replication. The observation may be exploitable for antiviral approaches.

DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions

Ig heavy chain (IgH) class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double-strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical nonhomologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining that is more biased to use longer junctional microhomologies (MHs). Deficiency for DSB response (DSBR) factors, including ataxia telangiectasia-mutated (ATM) and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B-lymphoma cells. Compared with wild-type, CSR and c-myc to S region translocation junctions in the absence of 53BP1, and, to a lesser extent, other DSBR factors, have increased MH utilization; indeed, 53BP1-deficient MH profiles resemble those associated with C-NHEJ deficiency. However, translocation junctions between c-myc DSB and general DSBs genome-wide are not MH-biased in ATM-deficient versus wild-type CH12F3 cells and are less biased in 53BP1- and C-NHEJ-deficient cells than CSR junctions or c-myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MH-biased end-joining in the absence of DSBR factors.

The RNA processing factors THRAP3 and BCLAF1 promote the DNA damage response through selective mRNA splicing and nuclear export.

mRNA splicing and export plays a key role in the regulation of gene expression, with recent evidence suggesting an additional layer of regulation of gene expression and cellular function through the selective splicing and export of genes within specific pathways. Here we describe a role for the RNA processing factors THRAP3 and BCLAF1 in the regulation of the cellular DNA damage response (DDR) pathway, a key pathway involved in the maintenance of genomic stability and the prevention of oncogenic transformation. We show that loss of THRAP3 and/or BCLAF1 leads to sensitivity to DNA damaging agents, defective DNA repair and genomic instability. Additionally, we demonstrate that this phenotype can be at least partially explained by the role of THRAP3 and BCLAF1 in the selective mRNA splicing and export of transcripts encoding key DDR proteins, including the ATM kinase. Moreover, we show that cancer associated mutations within THRAP3 result in deregulated processing of THRAP3/BCLAF1-regulated transcripts and consequently defective DNA repair. Taken together, these results suggest that THRAP3 and BCLAF1 mutant tumors may be promising targets for DNA damaging chemotherapy.

Abstract B14: A novel role for BRD9 in regulating cellular growth and DNA damage response pathways

Abstract Bromodomain containing protein BRD9 has been identified as a component of the SWI/SNF chromatin remodeling complex. Next generation sequencing studies have revealed that SWI/SNF is the most highly perturbed chromatin remodeling complex in cancer with subunit mutations present in approximately 20% of all tumor types. SWI/SNF has been implicated as both tumor suppressor and oncogenic driver in diverse contexts. For example, the catalytic subunit SMARCA4 functions as an oncogenic driver, essential for the maintenance of hematologic malignancies such as leukemia. In contrast, loss of function of the ATPase subunit SMARCA4 and the ARID1a subunit precipitate loss of tumor suppressor activity and are apparent in lung and ovarian subtypes. However, the role of BRD9 has not been clearly delineated. Clinical data indicates that testicular germ cell cancer (66%) and esophageal cancer (18%) bear a hemizygous deletion of BRD9 conferring HETLOSS status. Loss of BRD9 heterozygosity leading to enhanced tumorigenic potential is a hallmark of tumor suppressors. This highlights a novel role for this bromodomain protein in regulating the process of oncogenesis. Here we demonstrate shRNA-mediated knockdown of BRD9 enhances clonogenic potential of solid tumor cancer cell lines, while expression of wild-type BRD9 in a HETLOSS cell line abrogates colony formation. Collectively, our data suggests BRD9 functions as a regulator of cellular growth. We examined if SWI/SNF chromatin remodeling activity was dependent on BRD9 status and how this correlated with the growth phenotype. Nucleosome accessibility was significantly decreased when BRD9 was depleted indicating chromatin exists in a more compacted conformation. However, BRD9 knockdown does not result in alterations in assembly or stability of the SWI/SNF complex. Though loss of BRD9 does not perturb SWI/SNF dynamics, it may be required for complex recruitment to chromatin directly or by tethering through protein-protein interactions. Identification of novel BRD9-interacting proteins that target recruitment was carried out by proteomic survey. Analysis of the study revealed enrichment of proteins involved in DNA damage repair pathways. Using a double strand break reporter, we examined if BRD9 depletion perturbed homology-directed or non-homologous end joining repair pathways. Knockdown of BRD9 increased NHEJ-associated reporter activity but had subtle effect on homology-directed repair events. BRD9 colocalizes with γH2AX foci upon damage but this colocalization is lost upon depletion of BRD9. We propose that BRD9 is necessary for recruitment of SWI/SNF to sites of damage, to permit chromatin expansion and assembly of homology-directed repair factors. However, knockdown of BRD9 may shift the balance and favor interactions of non-homologous end joining pathway players to sites of damage, resulting in enhanced error-prone repair and ultimately leading to oncogenic transformation. Collectively, this work highlights a novel and previously unidentified role for BRD9 in DNA damage pathways and identifies a potential vulnerability in NHEJ dependence that may be therapeutically targeted. Citation Format: Caroline Vallaster, Farzin Gharadaghi, Alexis Cocozaki, Kelly Jacques, Brendan Price, Sylvie Guichard. A novel role for BRD9 in regulating cellular growth and DNA damage response pathways [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B14.

Abstract B37: PARP inhibitor olaparib induces genomic instability in normal mammalian cells

Abstract Poly(ADP-ribose) polymerases (PARPs) are the first proteins involved in cellular DNA damage response pathways to be targeted by specific inhibitors for clinical benefit. Tumors with defects in homologous recombination (HR) are hypersensitive to PARP inhibitors (PARPi), and early phase clinical trials have been promising in patients with advanced BRCA1 and BRCA2-associated breast, ovary and prostate cancer. Unlike HR-defective cells, HR-proficient cells manifest low cytotoxicity when exposed to PARPi. Nonetheless, they mount a DNA damage response and show a genomic instability phenotype as demonstrated by an increased frequency of sister chromatid exchange. To study molecular mechanisms underlying PARPi-generated genomic instability, we took advantage of olaparib an agent in clinical use. Our results in mouse embryonic stem cells show that olaparib increases HR leading to copy number alterations and induces a mutator phenotype. Further, using a genome wide approach we finally demonstrate that PARPi treatment increases DNA double-strand breaks genome-wide which can lead to translocations and other genome aberrations. Our findings have important implications for therapies with regard to sustained genotoxicity to normal cells. Genomic instability arising from PARPi warrants consideration in prevention strategies or for non-oncologic indications. Citation Format: Fabio Vanoli, Shuhei Ito, Richard L. Frock, Frederick W. Alt, Mary Ellen Moynahan, Maria Jasin. PARP inhibitor olaparib induces genomic instability in normal mammalian cells [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B37.

Inhibition of ATR in Combination with Nucleoside Analogues Eradicates Acute Myeloid Leukaemia in an Orthotopic Murine Xenograft Model

The ataxia telangiectasia and RAD3-related (ATR) protein kinase is a component of the cellular DNA damage response pathway and promotes cell survival by signalling repair of collapsed replication forks generated by replication stress. We hypothesised that inhibition of ATR potentiates the anti-leukaemic activity of chain terminating nucleoside analogues used in the treatment of acute myeloid leukaemia (AML). We used VE-821 and its derivative VX-970 (Vertex Pharmaceuticals, Abingdon, UK) as potent and specific inhibitors of ATR kinase activity to examine the effects of ATR inhibition in AML cell lines, primary AML cells and AML xenografts.Co-treatment with 1mM VE-821 did not consistently potentiate the anti-proliferative effects of cytarabine, clofarabine or fludarabine in a panel of AML cell lines. However, there was consistent potentiation of hydroxyurea and gemcitabine in all 7 AML cell lines tested. Treatment with hydroxyurea, which induces replication stress via depletion of dNTPs, resulted in phosphorylation of CHK1, a downstream target of ATR. CHK1 phosphorylation was attenuated when 1mM VE-821 was co-administered with hydroxyurea. Exposure of cells to gemcitabine or hydroxyurea slowed transit through S phase, which was pronounced in combination with VE-821. HL-60 AML cell clones expressing either a constitutively active or inducible shRNA construct targeting ATR had reduced ATR protein expression compared to control cells and were significantly more sensitive to the anti-proliferative effects of gemcitabine and hydroxyurea, but not to cytarabine, clofarabine or fludarabine.The growth inhibitory effects of hydroxyurea and gemcitabine were also significantly potentiated by VE-821 in primary AML patient samples, which included three adult patients with de novo AML and a paediatric patient with therapy-related AML. In contrast, ATR inhibition did not potentiate the inhibitory effects of hydroxyurea or gemcitabine in primary bone marrow cells from healthy donors ex vivo.We next sought to determine whether ATR inhibition potentiated hydroxyurea and gemcitabine in an orthotopic mouse model of AML. MV4-11 AML cells engineered to express firefly luciferase (MV4-11 pSLIEW) were intrafemorally transplanted into immunodeficient Rag2-/- gc-/- mice. Bioluminescent imaging via IVIS Spectrum (PerkinElmer, Buckinghamshire, UK) demonstrated localised femoral engraftment first detectable 4-5 days post-injection, with luciferase signal developing in other parts of the body (liver, ovaries) between days 15 and 18 in untreated mice. Treatment was initiated 7 days post-injection when disease was localised to the femur and prior to emergence of disseminated luciferase signal. Single agent hydroxyurea (250mg per kg, IP days 0-4 and 7-11) conferred some early disease control compared to controls as determined by luciferase total body flux measured on day 14, but this was not statistically significant (p=0.18) and did not affect overall survival (mean 35 days for controls and 37 days for hydroxyurea, p=0.47). Monotherapy with VX-970 (60 mg per kg, orally on days 0-4 and 7-11) also conferred early disease control compared to vehicle-treated mice (p=0.18), and resulted in significantly longer overall survival (mean 40 days, p=0.017). Combination treatment with hydroxyurea and VX-970 did not result in more effective early disease control or improved overall survival compared to monotherapy with either agent. Treatment with gemcitabine monotherapy (100 mg per kg, intraperitoneal injection on days 0, 3, 7 and 10) conferred significant early disease control (p=0.002) and significantly improved overall survival compared to controls (mean survival 73 days, p<0.001). Nevertheless, leukemic cells persisted and the cause of death in all mice randomised to this arm was relapsed disseminated disease. Treatment with gemcitabine and VX-970 in combination also conferred very significant early disease control (p=0.002), which eradicated luciferase signal by day 21 in all 6 mice randomised to this arm (Figure 1), and also conferred significantly improved overall survival compared to controls (p<0.001) or mice treated with gemcitabine monotherapy (p=0.001), with four mice remaining disease free at day 126. No significant weight loss or toxicity was observed in any of the treatment arms. Taken together, these data suggest ATR inhibition as a promising therapeutic strategy in AML. [Display omitted] DisclosuresPollard:Vertex Pharmaceuticals (Europe) Ltd.: Employment.

Epigenetic clock analyses of cellular senescence and ageing.

A confounding aspect of biological ageing is the nature and role of senescent cells. It is unclear whether the three major types of cellular senescence, namely replicative senescence, oncogene-induced senescence and DNA damage-induced senescence are descriptions of the same phenomenon instigated by different sources, or if each of these is distinct, and how they are associated with ageing. Recently, we devised an epigenetic clock with unprecedented accuracy and precision based on very specific DNA methylation changes that occur in function of age. Using primary cells, telomerase-expressing cells and oncogene-expressing cells of the same genetic background, we show that induction of replicative senescence (RS) and oncogene-induced senescence (OIS) are accompanied by ageing of the cell. However, senescence induced by DNA damage is not, even though RS and OIS activate the cellular DNA damage response pathway, highlighting the independence of senescence from cellular ageing. Consistent with this, we observed that telomerase-immortalised cells aged in culture without having been treated with any senescence inducers or DNA-damaging agents, re-affirming the independence of the process of ageing from telomeres and senescence. Collectively, our results reveal that cellular ageing is distinct from cellular senescence and independent of DNA damage response and telomere length.

Abstract PL04-02: ATM: Bridging DNA damage responses and metabolic regulation

Abstract Much of our understanding of the mechanisms involved in cellular DNA damage response pathways have come from studies of human cancer susceptibility syndromes. ATM, the gene mutated in the cancer-prone, radiosensitive disorder, Ataxia-telangiectasia (A-T), is a protein kinase that is a central mediator of responses to DNA double strand breaks in cells. Once activated, ATM phosphorylates numerous substrates in the cell that modulate the cell's response to the DNA damage. p53, one of the many targets of the ATM kinase, is a critical mediator of cell cycle changes and cell death signaling following DNA damage and other stresses. Though ATM clearly plays a central role in mediating cellular responses to DNA breakage, some of the clinical abnormalities in A-T are difficult to attribute directly to alterations in DNA damage signaling. The broad-based symptoms in A-T and recently acquired data suggest that ATM has physiologic roles that extend beyond its regulation of DNA damage responses. In particular, ATM appears to affect insulin signaling pathways and to have a direct role in regulating mitochondrial function. Interestingly, we found in mouse models of A-T that these mitochondrial defects associated with ATM deficiency appear to be a contributing factor to cancer development in A-T. These new insights suggest that Ataxia-Telangiectasia should be considered, at least in part, as a mitochondrial dysfunction disease and provide links between cancer-related DNA damage signaling, general metabolic signaling pathways, and cancer development. Citation Format: Michael B. Kastan, Yasmine Valentin-Vega, Adam Brown, Peter Scarbrough, Donald Fleenor. ATM: Bridging DNA damage responses and metabolic regulation. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PL04-02.

HSV-I and the cellular DNA damage response.

Peter Wildy first observed genetic recombination between strains of HSV in 1955. At the time, knowledge of DNA repair mechanisms was limited, and it has only been in the last decade that particular DNA damage response (DDR) pathways have been examined in the context of viral infections. One of the first reports addressing the interaction between a cellular DDR protein and HSV-1 was the observation by Lees-Miller et al. that DNA-dependent protein kinase catalytic subunit levels were depleted in an ICP0-dependent manner during Herpes simplex virus 1 infection. Since then, there have been numerous reports describing the interactions between HSV infection and cellular DDR pathways. Due to space limitations, this review will focus predominantly on the most recent observations regarding how HSV navigates a potentially hostile environment to replicate its genome.

Checkpoint kinase 1 is activated and promotes cell survival after exposure to sulphur mustard

Sulphur mustard (SM) is a vesicating agent that has been used several times as a weapon during military conflict and continues to pose a threat as an agent of warfare/terrorism. After exposure, SM exerts both acute and delayed long-term toxic effects principally to the skin, eyes and respiratory system. These effects are thought to be mediated, at least in part, by direct interaction of SM with DNA, forming a myriad of DNA lesions and initiating effects on cell cycle and cell death pathways. Previous studies have demonstrated that a complex network of cellular DNA damage response pathways are utilised in cells exposed to SM, consistent with SM causing multiple forms of DNA damage. The present study focused on the role of Checkpoint kinase 1 (CHK1), a protein with putative roles in homologous recombination repair, p53 activation and the initiation of cell cycle checkpoints after certain forms of DNA damage. The data showed that SM caused robust activation of CHK1, monitored by multi-site phosphorylation analysis and that this activation was dependent on the ataxia telangiectasia and Rad3-related (ATR) protein kinase. Furthermore, specific inhibition of CHK1 increased SM toxicity in multiple human cell lines, with concomitant increases in markers of apoptosis, DNA damage and mitosis. Finally, the effect of CHK1 inhibition on SM toxicity was much more marked in cells with non-functional p53.

Tools and strategies for DNA damage interactome analysis

DNA is the target of multiple endogenous and exogenous agents generating chemical lesions on the double helix. Cellular DNA damage response pathways rely on a myriad of proteins interacting with DNA alterations. The cartography of this interactome currently includes well known actors of chromatin remodelling, DNA repair or proteins hijacked from their natural functions such as transcription factors. In order to go further into the characterisation of these protein networks, proteomics-based methods began to be used in the early 2000s. The strategies are diverse and include mainly (i) damaged DNA molecules used as targets on protein microarrays, (ii) damaged DNA probes used to trap within complex cellular extracts proteins that are then separated and identified by proteomics, (iii) identification of chromatin- bound proteins after a genotoxic stress, or (iv) identification of proteins associated with other proteins already known to be part of DNA damage interactome. All these approaches have already been performed to find new proteins recognizing oxidised bases, abasic sites, strand breaks or crosslinks generated by anticancer drugs such as nitrogen mustards and platinating agents. Identified interactions are generally confirmed using complementary methods such as electromobility shift assays or surface plasmon resonance. These strategies allowed, for example, demonstration of interactions between cisplatin-DNA crosslinks and PARP-1 or the protein complex PTW/PP. The next challenging step will be to understand the biological repercussions of these newly identified interactions which may help to unravel new mechanisms involved in genetic toxicology, discover new cellular responses to anticancer drugs or identify new biomarkers and therapeutic targets.

Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA

Adenoviruses are linear double stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor since mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.