ATR Pathway Activation Research Articles

Reducing DNA Damage Associated with Endogenous Genotoxic Stress in Hematopoietic Stem Cells

Fanconi anemia (FA) is a bone marrow failure (BMF) syndrome that arises from mutations in a network of FA genes essential for DNA interstrand crosslink (ICL) repair and replication stress tolerance. Endogenous formaldehyde is ubiquitous within cells as it is produced as a byproduct during normal cellular metabolism such as histone, RNA and DNA demethylation. Previous studies show that endogenous formaldehyde is a hematopoietic stem cell (HSC) genotoxin and metabolic carcinogen. We previously showed that endogenous formaldehyde is removed by the enzyme alcohol dehydrogenase 5 (ADH5), and Adh5-deficient mice therefore accumulate formaldehyde adducts in DNA that require the FA pathway to repair. Mice lacking Adh5 together with the FA pathway central component Fancd2 exhibit bone marrow failure and early lethality. Adh5 haploinsufficiency combined with Fancd2 deficiency, or Fancd2 -/- Adh5 +/- mice, demonstrates severely compromised HSC activity. However, the contributing factors that cause DNA damage and the protective mechanisms by which cells reduce DNA damage during endogenous genotoxic stress are not fully understood. Here, we show that the genetic inactivation of Lnk/Sh2b3 in Fancd2 -/- Adh5 +/- mice restores HSC function. Adh5 haploinsufficiency exacerbated Fancd2 deficiency, while Lnk deficiency rescued HSC homeostasis and transplantability of Fancd2 -/- Adh5 +/- mice. Moreover, Lnk deficiency increased HSC quiescence and reduced endogenous DNA damage in Fancd2 -/- Adh5 +/- mice. Importantly, Lnk deficiency does not participate in repairing formaldehyde-induced DNA damage, nor does it metabolize formaldehyde or reduce genomic formaldehyde-deoxyguanine adduct in vivo. Rather, Lnk deficiency reduced single-strand DNA (ssDNA) breaks and ssDNA binding protein RPA in HSCs during the cell cycle, thereby suppressing the ATR pathway activation upon endogenous replication stress. Moreover, loss of Lnk increased S-phase progression and fork recovery in Fancd2 -/- Adh5 +/- HSPCs at the stressed replication fork upon HU treatment, thereby preventing replication fork collapse and DNA damage. These findings highlight a new mechanism by which Lnk deficiency mitigates replication stress-induced DNA damage in response to endogenous genotoxic insult from cellular metabolism during HSC regeneration.

Enhancer-driven transcription of MCM8 by E2F4 promotes ATR pathway activation and glioma stem cell characteristics

BackgroundGlioma stem cells (GSCs) are responsible for glioma recurrence and drug resistance, yet the mechanisms underlying their maintenance remains unclear. This study aimed to identify enhancer-controlled genes involved in GSCs maintenance and elucidate the mechanisms underlying their regulation.MethodsWe analyzed RNA-seq data and H3K27ac ChIP-seq data from GSE119776 to identify differentially expressed genes and enhancers, respectively. Gene Ontology analysis was performed for functional enrichment. Transcription factors were predicted using the Toolkit for Cistrome Data Browser. Prognostic analysis and gene expression correlation was conducted using the Chinese Glioma Genome Atlas (CGGA) data. Two GSC cell lines, GSC-A172 and GSC-U138MG, were isolated from A172 and U138MG cell lines. qRT-PCR was used to detect gene transcription levels. ChIP-qPCR was used to detect H3K27ac of enhancers, and binding of E2F4 to target gene enhancers. Western blot was used to analyze protein levels of p-ATR and γH2AX. Sphere formation, limiting dilution and cell growth assays were used to analyze GSCs growth and self-renewal.ResultsWe found that upregulated genes in GSCs were associated with ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) pathway activation, and that seven enhancer-controlled genes related to ATR pathway activation (LIN9, MCM8, CEP72, POLA1, DBF4, NDE1, and CDKN2C) were identified. Expression of these genes corresponded to poor prognosis in glioma patients. E2F4 was identified as a transcription factor that regulates enhancer-controlled genes related to the ATR pathway activation, with MCM8 having the highest hazard ratio among genes positively correlated with E2F4 expression. E2F4 bound to MCM8 enhancers to promote its transcription. Overexpression of MCM8 partially restored the inhibition of GSCs self-renewal, cell growth, and the ATR pathway activation caused by E2F4 knockdown.ConclusionOur study demonstrated that E2F4-mediated enhancer activation of MCM8 promotes the ATR pathway activation and GSCs characteristics. These findings offer promising targets for the development of new therapies for gliomas.

Abstract IA03: Polyploidy, DNA damage response driver or gatekeeper of chronic liver diseases

Abstract Polyploidy, also known as whole-genome amplification, is a condition in which the organism has more than the two basic sets of chromosomes. Polyploidy frequently arises during tissue development and repair, and in age-associated diseases, such as cancer. Its consequences are diverse and clearly different between systems. The liver is a particularly fascinating organ in that it can adapt its ploidy to the physiological and pathological context. In human liver parenchyma, polyploidy is mainly cellular (binuclear hepatocytes) with no specific zonation across the liver lobule. During liver tumorigenesis, cellular ploidy is drastically reduced; binuclear polyploid hepatocytes are barely present in HCC tumors. Remarkably, nuclear ploidy is specifically amplified in HCC tumors. In fact, nuclear ploidy is amplified in HCCs harboring a low degree of differentiation and TP53 mutations. Highly polyploid tumors are associated with a poor prognosis. Hepatocytes increase their ploidy also during the proliferative stress that underlies various liver disease conditions. Recently, we investigated mechanisms underlying activation of the DNA damage response during Non-alcoholic Steatotic Liver Disease. We demonstrated that proliferating mouse NAFLD hepatocytes harbor replication stress with an alteration of the replication fork’s speed and activation of ATR pathway which is sufficient to cause DNA breaks. Nucleotide pool imbalance occurring during NAFLD is the key driver of replication stress. Remarkably, DNA lesions drive cGAS/STING pathway activation, a major component of cells’ intrinsic immune response. The translational significance of this study was reiterated by showing that lipid overload in proliferating HepaRG was sufficient to induce replication stress and nucleotide pool imbalance. Moreover, livers from NAFLD human patients displayed nucleotide pathways deregulation and cGAS/STING genes alteration. Altogether, our findings shed new light on the mechanisms by which damaged NAFLD hepatocytes might promote disease progression. Citation Format: Chantal Desdouets. Polyploidy, DNA damage response driver or gatekeeper of chronic liver diseases [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr IA03.

Replication stress triggered by nucleotide pool imbalance drives DNA damage and cGAS-STING pathway activation in NAFLD.

Non-alcoholic steatotic liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. NAFLD has a major effect on the intrinsic proliferative properties of hepatocytes. Here, we investigated the mechanisms underlying the activation of DNA damage response during NAFLD. Proliferating mouse NAFLD hepatocytes harbor replication stress (RS) with an alteration of the replication fork's speed and activation of ATR pathway, which is sufficient to cause DNA breaks. Nucleotide pool imbalance occurring during NAFLD is the key driver of RS. Remarkably, DNA lesions drive cGAS/STING pathway activation, a major component of cells' intrinsic immune response. The translational significance of this study was reiterated by showing that lipid overload in proliferating HepaRG was sufficient to induce RS and nucleotide pool imbalance. Moreover, livers from NAFLD patients displayed nucleotide pathway deregulation and cGAS/STING gene alteration. Altogether, our findings shed light on the mechanisms by which damaged NAFLD hepatocytes might promote disease progression.

ADAR1 Prevents R-loop Accumulation-Driven ATR Pathway Activation in Ovarian Cancer.

Adenosine (A)-to-inosine (I) RNA editing is the most prevalent RNA editing mechanism, in which adenosine deaminase acting on RNA 1 (ADAR1) is a major adenosine deaminase. Increasing evidence suggests that editing dysregulation of ADAR1 plays an important role in human tumorigenesis, while the underlying mechanism remains elusive. Here, we demonstrated that ADAR1 was highly expressed in ovarian cancer tissues and negatively correlated with progression free survival of ovarian cancer patients. Importantly, silence of ADAR1 repressed ovarian cancer cell growth and colony formation in vitro and inhibited ovarian cancer cell tumorigenesis in vivo. Further cell cycle and transcriptome profile analysis revealed that silence of ADAR1 in ovarian cancer cells induced cell cycle arrest at G1/G0 stage. Mechanistically, loss of ADAR1 caused R-loop abnormal accumulation, thereby contributing to single stand DNA break and ATR pathway activation. Additionally, ADAR1 interacted with DHX9 to regulate R-loop complex formation, and A-to-I editing of nascent RNA repressed R-loop formation during co-transcriptional process. Together, our results identify a novel ADAR1/R-loop/ATR axis critical for ovarian cancer progression and a potential target for ovarian cancer therapy.

TOP1 inhibition induces bifurcated JNK/MYC signaling that dictates cancer cell sensitivity.

Rationale: Triple-negative breast cancer (TNBC) does not respond to anti-estrogen and anti-HER2 therapies and is commonly treated by chemotherapy. TNBC has a high recurrence rate, particularly within the first 3 years. Thus, there is an urgent clinical need to develop more effective therapies for TNBC. Topoisomerase I (TOP1) inhibitors cause DNA damage, making these drugs desirable for TNBC treatment since DNA repair machinery is defective in this subtype of breast cancer. Among the main molecular subtypes of breast cancer, the TNBC cell lines exhibited the highest TOP1 inhibition sensitivity. However, clinically used TOP1 inhibitors, such as topotecan and irinotecan, have shown limited clinical applications and the reasons remain unclear. Understanding the mechanism of differential responses to TOP1 blockade and identifying the predictive markers for cancer cell sensitivity will help further TOP1-targeted therapy for TNBC treatment and improve the clinical use of TOP1 inhibitors.Methods: Viability assays were used to evaluate breast cancer cell sensitivity to topotecan and other TOP1 inhibitors as well as TOP2 inhibitors. An in vitro-derived topotecan-resistant TNBC cell model and TNBC xenograft models were employed to confirm cancer cell response to TOP1 blockade. RNA-seq was used to identify potential predictive markers for TNBC cell response to TOP1 blockade. Western blotting and qRT-PCR were performed to measure the protein levels and RNA expression. ATAC-seq and luciferase reporter assays were used to examine MYC transcriptional regulations. The effects of MYC and JNK in cancer cell response to TOP1 inhibition were validated via loss-of-function and gain-of-function experiments.Results: We observed two distinct and diverging cancer cell responses - sensitive versus resistant to TOP1 inhibition, which was confirmed by TNBC xenograft mouse models treated by topotecan. TNBC cells exhibited bifurcated temporal patterns of ATR pathway activation upon TOP1 inhibitor treatment. The sensitive TNBC cells showed an “up then down” dynamic pattern of ATR/Chk1 signaling, while the resistant TNBC cells exhibited a “persistently up” profile. On the contrary, opposite temporal patterns of induced expression of MYC, a key regulator and effector of DNA damage, were found in TNBC cells treated by TOP1 inhibitors. Mechanistically, we showed that TOP1-induced JNK signaling upregulated MYC expression. Furthermore, pharmacological inhibition of ATR reversed TNBC cell resistance to topotecan, whereas MYC knockdown and JNK inhibition reduced cancer cell sensitivity.Conclusions: Dynamic temporal profiles of induced ATR/Chk1 and JNK activation as well as MYC expression, may predict cancer cell response to TOP1 inhibitors. JNK activation-mediated constitutive elevation of MYC expression may represent a novel mechanism governing cancer cell sensitivity to TOP1-targeting therapy. Our results may provide implications for identifying TNBC patients who might benefit from the treatment with TOP1 inhibitors.

ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses.

Previously, we reported that cellular transcription factor ZASC1 facilitates DNA-dependent/RNA-independent recruitment of HIV-1 TAT and the cellular elongation factor P-TEFb to the HIV-1 promoter and is a critical factor in regulating HIV-1 transcriptional elongation (PLoS Path e1003712). Here we report that cellular transcription factor ZBTB2 is a novel repressor of HIV-1 gene expression. ZBTB2 strongly co-immunoprecipitated with ZASC1 and was dramatically relocalized by ZASC1 from the cytoplasm to the nucleus. Mutations abolishing ZASC1/ZBTB2 interaction prevented ZBTB2 nuclear relocalization. We show that ZBTB2-induced repression depends on interaction of cellular histone deacetylases (HDACs) with the ZBTB2 POZ domain. Further, ZASC1 interaction specifically recruited ZBTB2 to the HIV-1 promoter, resulting in histone deacetylation and transcription repression. Depleting ZBTB2 by siRNA knockdown or CRISPR/CAS9 knockout in T cell lines enhanced transcription from HIV-1 vectors lacking Vpr, but not from these vectors expressing Vpr. Since HIV-1 Vpr activates the viral LTR by inducing the ATR kinase/DNA damage response pathway, we investigated ZBTB2 response to Vpr and DNA damaging agents. Expressing Vpr or stimulating the ATR pathway with DNA damaging agents impaired ZASC1’s ability to localize ZBTB2 to the nucleus. Moreover, the effects of DNA damaging agents and Vpr on ZBTB2 localization could be blocked by ATR kinase inhibitors. Critically, Vpr and DNA damaging agents decreased ZBTB2 binding to the HIV-1 promoter and increased promoter histone acetylation. Thus, ZBTB2 is recruited to the HIV-1 promoter by ZASC1 and represses transcription, but ATR pathway activation leads to ZBTB2 removal from the promoter, cytoplasmic sequestration and activation of viral transcription. Together, our data show that ZASC1/ZBTB2 integrate the functions of TAT and Vpr to maximize HIV-1 gene expression.

The POLD1R689W variant increases the sensitivity of colorectal cancer cells to ATR and CHK1 inhibitors

Inhibition of the kinase ATR, a central regulator of the DNA damage response, eliminates subsets of cancer cells in certain tumors. As previously shown, this is at least partly attributable to synthetic lethal interactions between ATR and POLD1, the catalytic subunit of the polymerase δ. Various POLD1 variants have been found in colorectal cancer, but their significance as therapeutic targets for ATR pathway inhibition remains unknown. Using CRISPR/Cas9 in the colorectal cancer cell line DLD-1, which harbors four POLD1 variants, we established heterozygous POLD1-knockout clones with exclusive expression of distinct variants to determine the functional relevance of these variants individually by assessing their impact on ATR pathway activation, DNA replication, and cellular sensitivity to inhibition of ATR or its effector kinase CHK1. Of the four variants analyzed, only POLD1R689W affected POLD1 function, as demonstrated by compensatory ATR pathway activation and impaired DNA replication. Upon treatment with ATR or CHK1 inhibitors, POLD1R689W strongly decreased cell survival in vitro, which was attributable at least partly to S phase impairment and apoptosis. Similarly, treatment with the ATR inhibitor AZD6738 inhibited growth of murine xenograft tumors, harboring the POLD1R689W variant, in vivo. Our POLD1-knockout model thus complements algorithm-based models to predict the pathogenicity of tumor-specific variants of unknown significance and illustrates a novel and potentially clinically relevant therapeutic approach using ATR/CHK1 inhibitors in POLD1-deficient tumors.

Replication stress signaling is a therapeutic target in myelodysplastic syndromes with splicing factor mutations.

Somatic mutations in genes coding for splicing factors, e.g., SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with myelodysplastic syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease, therefore, making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation- associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with the low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association between R-loops and ATR sensitivity and the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.

The SF3B1 K700E Mutation Induces R-Loop Accumulation and Associated DNA Damage

The myelodysplastic syndromes (MDS) are common myeloid malignancies. Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1 and ZRSR2) are the most common mutations found in MDS. There is evidence that some spliceosomal components play a role in the maintenance of genomic stability. Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of R-loops (RNA-DNA hybrids with a displaced single stranded DNA). Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loops formation in leukemia cell lines, resulting in increased DNA damage, replication stress and activation of the ATR-Chk1 pathway. SF3B1 is the most frequently mutated splicing factor gene in MDS, but a role for mutated SF3B1 in R-loop accumulation and DNA damage has not yet been reported in hematopoietic cells. We have investigated the effects of the common SF3B1 K700E mutation on R-loop formation and DNA damage response in MDS and leukemia cells. R-loop signals and the DNA damage response were measured by immunofluorescence staining using S9.6 and anti-γ-H2AX antibodies respectively. Firstly, we studied K562 (myeloid leukemia) cells with the SF3B1 K700E mutation and isogenic SF3B1 K700K wildtype (WT) K562 cells. K562 cells with SF3B1 mutation showed a significant increase in the number of S9.6 foci [Fold change (FC) 2.01, p<0.001] and in the number of γ-H2AX foci (FC 2.32, p<0.001), indicating increased R-loops and DNA damage, compared to SF3B1 WT K562 cells. Moreover, we observed increased Chk1 phosphorylation at Ser345, a hallmark of activation of the ATR pathway, in SF3B1 mutant K562 cells. Next, we analyzed induced pluripotent stem cells (iPSCs) that we generated from the bone marrow cells of one SF3B1 mutant MDS patient and of one healthy control. A significant increase in R-loops and DNA damage response was observed in an iPSC clone harboring SF3B1 mutation compared to another iPSC clone without SF3B1 mutation obtained from same MDS patient (S9.6 mean fluorescence intensity - FC 1.72, p<0.001; γ-H2AX foci - FC 1.34, p=0.052) and to iPSCs from the healthy control (S9.6 mean fluorescence intensity - FC 1.53, p<0.001; γ-H2AX foci - FC 1.61, p=0.006). In addition, bone marrow CD34+ cells from a SF3B1 mutant MDS patient showed increased R-loops (as measured by number of S9.6 foci) compared to CD34+ cells from a MDS patient without splicing factor mutations (FC 1.9) and from a healthy control (FC 2.6). To investigate whether the observed DNA damage and ATR activation in SF3B1 mutant K562 cells result from induced R-loops, we overexpressed RNASEH1 (encoding an enzyme that degrades the RNA in RNA:DNA hybrids) to resolve R-loops in these cells. RNASEH1 overexpression significantly reduced the number of S9.6 (FC 0.51, p<0.001) and γ-H2AX foci (FC 0.63, p=0.035) in SF3B1 mutant K562 cells compared to SF3B1 WT K562 cells. RNASEH1 overexpression also resulted in decreased Chk1 phosphorylation, indicating suppression of ATR pathway activation in SF3B1 mutant K562 cells. To determine the functional importance of ATR activation associated with SF3B1 mutation, we evaluated the sensitivity of SF3B1 mutant cells towards the ATR inhibitor VE-821. SF3B1 mutant K562 cells showed preferential sensitivity towards VE-821 compared to SF3B1 WT K562 cells. Chk1 is a critical substrate of ATR, and we next investigated the effects of Chk1 inhibition in SF3B1 mutant cells. Interestingly, SF3B1 mutant K562 cells demonstrated preferential sensitivity towards the Chk1 inhibitor UCN-1 (IC50 61.8 nM) compared to SF3B1 WT K562 cells (IC50 267 nM), suggesting that ATR activation is important for the survival of SF3B1 mutant cells. SF3B1 mutant K562 cells were preferentially sensitive to the splicing modulator Sudemycin D6 (IC50 53.2 nM) compared to SF3B1 WT K562 cells (IC50 130.7 nM). The effects of VE-821 and UCN-1 on SF3B1 mutant K562 cells were enhanced by Sudemycin D6 (Combination index <1), indicating synergy. In summary, our results show that the SF3B1 mutation leads to accumulation of R-loops and associated DNA damage resulting in activation of the ATR pathway in MDS and leukemia cells. Thus different mutated splicing factors have convergent effects on R-loop elevation leading to DNA damage. Moreover, our data suggest that Chk1 inhibition, alone or in combination with splicing modulators, may represent a novel therapeutic strategy to target SF3B1 mutant cells. Disclosures Schuh: Janssen: Speakers Bureau; Verastem: Speakers Bureau; Kite: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau. Wiseman:Novartis, Celgene: Consultancy, Honoraria.

Abstract 4286: Tissue-specific preference for ATM or ATR pathway activation determines DNA damage outcome and provides insights in cancer predisposition

Abstract The DNA damage response (DDR) is an elaborate signaling network that protects against mutations and genomic instability. Despite its importance in preventing carcinogenesis, certain mutations in the DDR (e.g. BRCA1/2) predispose for specific cancers only. An explanation for this could be that the DDR is regulated differently across tissues; however, systematic analyses of the response to DNA damage in healthy human cells have not been carried out. We analyzed proteomic changes in response to single- and double strand DNA damage in non-immortalized human primary cells and related them to cell outcome. Primary breast and lung cells were treated with cisplatin (single-strand damage) or doxorubicin (double-strand breaks) at doses that caused a similar amount of DNA damage. Cell outcomes that were studied were apoptosis and cell cycle progression. Protein dynamics of 366 proteins were assessed using protein arrays and quantitative western blots. Inhibitors and an ODE-based mathematical model were used to determine the network topology and link it to outcome. Treatment of primary breast and lung epithelial cells with an equal amount of damage resulted in different viabilities. Primary mammary cells showed higher levels of apoptosis after single-strand damage, primary airway cells were more sensitive to double-strand breaks. The difference in outcome was reflected in distinct protein dynamics, including DNA repair pathways. Surprisingly, these differences originated at the earliest level of DNA damage detection by ATM and ATR. We demonstrated that differential activation of ATM and ATR pathways in breast and lung cells affects p53 and p21 dynamics and leads to distinct outcomes. We were able to identify key network nodes, providing new insights into the regulation of p53 and p21 and their relation to cell outcome. p53 and p21 dynamics are also linked to regulation of the cell cycle. In line with this, we found that cell cycle progression under normal conditions and after DNA damage is different. Importantly, we show that the double-strand break pathway plays a larger role in cell cycle control in breast cells than in lung cells. Since cell cycle checkpoints are a safeguard against genomic instability, differential regulation may affect cancer predisposition in a cell-type specific manner. Our work may provide an explanation for the fact that certain mutations in the DSB-repair pathway predispose for breast cancer, but not for lung cancer. In summary, we show that primary breast and lung cells activate distinct programs in response to equal doses of DNA damage, resulting in different p53 and p21 dynamics and, consequently, diverse outcomes. In addition, this work sheds light on the tissue-specific cancer predisposition of mutated DNA repair genes and may be the basis for further studies. Citation Format: Marijn T. Van Jaarsveld, Difan Deng, Erik A. Wiemer, Zhike Zi. Tissue-specific preference for ATM or ATR pathway activation determines DNA damage outcome and provides insights in cancer predisposition [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 4286.

Ronin influences the DNA damage response in pluripotent stem cells

Early mammalian embryonic cells must maintain a particularly robust DNA repair system, as mutations at this developmental point have detrimental consequences for the organism. How the repair system can be tuned to fulfill such elevated requirements is largely unknown, but it may involve transcriptional regulation. Ronin (Thap11) is a transcriptional regulator responsible for vital programs in pluripotent cells. Here, we report that this protein also modulates the DNA damage response of such cells. We show that conditional Ronin knockout sensitizes embryonic stem cells (ESCs) to UV-C-induced DNA damage in association with Atr pathway activation and G2/M arrest. Ronin binds to and regulates the genes encoding several DNA repair factors, including Gtf2h4 and Rad18, providing a potential mechanism for this phenotype. Our results suggest that the unique DNA repair requirements of the early embryo are not met by a static system, but rather via highly regulated processes.

Cytoplasmic ATR Activation Promotes Vaccinia Virus Genome Replication

SummaryIn contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

Small-molecule inhibitors of Ataxia Telangiectasia and Rad3 related kinase (ATR) sensitize lymphoma cells to UVA radiation

BackgroundPsoralen plus ultraviolet A (PUVA) photochemotherapy is a combination treatment used for inflammatory and neoplastic skin diseases such as mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL). However, 30% of MF patients do not respond sufficiently to PUVA and require more aggressive therapies. ObjectiveThe aim of this project was to investigate whether inhibition of Ataxia Telangiectasia and Rad3 related kinase (ATR) may enhance efficacy of phototherapy. MethodsCTCL cell lines (MyLa2000, SeAx and Mac2a) served as in vitro cell models. ATR and Chk1 were inhibited by small molecule antagonists VE-821, VE-822 or Chir-124, or by small interfering RNAs (siRNAs). Cell cycle and viability were assessed by flow cytometry. ResultsSmall molecule inhibitors of ATR and Chk1 potently sensitized all cell lines to PUVA and, importantly, also to UVA, which by itself did not cause apoptotic response. VE-821/2 blocked ATR pathway activation and released the cells from the G2/M block caused by UVA and PUVA, but did not affect apoptosis caused by other chemotherapeutics (etoposide, gemcitabine, doxorubicine) or by hydrogen peroxide. Knockdown of ATR and Chk1 with siRNA also blocked the ATR pathway and released the cells from G2/M block but did not sensitize the cells to UVA as observed with the small molecule inhibitors. The latter suggested that the synergism between VE-821/2 or Chir-124 and UVA was not solely caused by specific blocking of ATR kinase but also ATR-independent photosensitization. This hypothesis was further verified by administrating VE-821/2 or Chir-124 before and after UVA irradiation, as well as comparing their activity with other ATR and Chk1 inhibitors (AZD6738 and MK8776). We found that only VE-821/2 and Chir-124 kinase inhibitors had synergistic effect with UVA, and only if applied before treatment with UVA. ConclusionSmall molecule ATR and Chk1 inhibitors potently sensitize lymphoma cells to UVA radiation and induce a prominent apoptotic response. Interestingly, this effect is due to the dual (kinase inhibiting and photosensitizing) mode of action of these compounds.

The complexity of DNA double strand break is a crucial factor for activating ATR signaling pathway for G2/M checkpoint regulation regardless of ATM function.

DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation.

Tim–Tipin dysfunction creates an indispensible reliance on the ATR–Chk1 pathway for continued DNA synthesis

The Tim (Timeless)-Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim-Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR-Chk1 pathway engagement. We now demonstrate that Tim-Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR-Chk1 pathway in Tim-Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim-Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim-Tipin dysfunction. Together, these experiments indicate that the Tim-Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR-Chk1 function and by facilitating phosphorylation of Chk1 by ATR.