ATR-Chk1 Checkpoint Research Articles

Depletion of histone H3K27 demethylase exerts anti-tumor activity as a monotherapy and in combination therapy with ceralasertib in non-small cell lung carcinoma.

e20035 Background: Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide. Non-small cell lung carcinoma (NSCLC) represents 85% of lung cancers and is the most common type with poor prognosis. The lysine-specific demethylase UTX (gene name KDM6A) demethylates H3K27me2/3 at genes and enhancers and have been shown to support oncogenic transcription factors. We hypothesize that UTX is a highly pleiotropic factor in tumorigenesis and may plays a critical role in controlling DNA replication-associated gene activation as well as in replication stress-related DNA damage repair. Methods: To evaluate the role of UTX in tumorigenesis, shRNA-mediated UTX knockdown was employed in NCI-H1975 cells followed by cell cycle and cell proliferation assays. In the UTX-knocked down NCI-H1975 cells, target genes involved in DNA replication and DNA damage repair were discovered by RNA sequencing and further validated with RT-qPCR. Effects of UTX depletion on DNA replication and radiation-induced DNA repair were analyzed by immunofluorescence. Effects of UTX depletion on cell cycle arrest and the ATR–CHK1 checkpoint signaling pathway were analyzed by flow cytometry and immunoblotting. In vivo response to UTX inhibition monotherapy (UTX knockdown) and combination therapy of UTX knockdown with ceralasertib, a selective ATR kinase inhibitor, was evaluated in dox-inducible UTX knockdown NCI-H1975 xenografts. Results: Induction of UTX knockdown significantly reduced the NCI-H1975 tumor volume compared to the control group, showing anti-tumor activity. Furthermore, loss of UTX induced hyperactivation of ATR–CHK1 checkpoint signaling pathway, suggesting the UTX-depleted tumor cells attempt to alleviate replication stress to escape from apoptosis. To inhibit the UTX-depletion induced ATR–CHK1 pathway, ceralasertib (formerly AZD6738), an oral potent selective inhibitor of ATR, was combined with UTX-knockdown for in vitro and in vivo evaluation. Consistent with in vitro cell-based assay, combination therapy of ceralasertib with UTX depletion showed enhanced tumor growth inhibition and regression in the NCI-H1975 xenograft model. Conclusions: Our findings provide new insights into a pro-oncogenic role of UTX in supporting tumor maintenance via engaging DNA replication and repair pathway. Depletion of UTX alone may serve as a new therapeutic target in non-small cell lung carcinoma; and further combination with ceralasertib enhances the anti-tumor activity of UTX knockdown by inhibiting the ATR–CHK1 checkpoint signaling pathway.

The Novel ATR Inhibitor M1774 Induces Replication Protein Overexpression and Broad Synergy with DNA-targeted Anticancer Drugs.

Ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase inhibitors are in clinical trials. Here we explored the molecular pharmacology and therapeutic combination strategies of the oral ATR inhibitor M1774 (Tuvusertib) with DNA-damaging agents (DDA). As single agent, M1774 suppressed cancer cell viability at nanomolar concentrations, showing greater activity than ceralasertib and berzosertib, but less potency than gartisertib and elimusertib in the small cell lung cancer H146, H82, and DMS114 cell lines. M1774 also efficiently blocked the activation of the ATR-CHK1 checkpoint pathway caused by replication stress induced by TOP1 inhibitors. Combination with non-toxic dose of M1774 enhanced TOP1 inhibitor-induced cancer cell death by enabling unscheduled replication upon replicative damage, thereby increasing genome instability. Tandem mass tag-based quantitative proteomics uncovered that M1774, in the presence of DDA, forces the expression of proteins activating replication (CDC45) and G2-M progression (PLK1 and CCNB1). In particular, the fork protection complex proteins (TIMELESS and TIPIN) were enriched. Low dose of M1774 was found highly synergistic with a broad spectrum of clinical DDAs including TOP1 inhibitors (SN-38/irinotecan, topotecan, exatecan, and exatecan), the TOP2 inhibitor etoposide, cisplatin, the RNA polymerase II inhibitor lurbinectedin, and the PARP inhibitor talazoparib in various models including cancer cell lines, patient-derived organoids, and mouse xenograft models. Furthermore, we demonstrate that M1774 reverses chemoresistance to anticancer DDAs in cancer cells lacking SLFN11 expression, suggesting that SLFN11 can be utilized for patient selection in upcoming clinical trials.

Replisome dysfunction upon inducible TIMELESS degradation synergizes with ATR inhibition to trigger replication catastrophe.

The structure of DNA replication forks is preserved by TIMELESS (TIM) in the fork protection complex (FPC) to support seamless fork progression. While the scaffolding role of the FPC to couple the replisome activity is much appreciated, the detailed mechanism whereby inherent replication fork damage is sensed and counteracted during DNA replication remains largely elusive. Here, we implemented an auxin-based degron system that rapidly triggers inducible proteolysis of TIM as a source of endogenous DNA replication stress and replisome dysfunction to dissect the signaling events that unfold at stalled forks. We demonstrate that acute TIM degradation activates the ATR-CHK1 checkpoint, whose inhibition culminates in replication catastrophe by single-stranded DNA accumulation and RPA exhaustion. Mechanistically, unrestrained replisome uncoupling, excessive origin firing, and aberrant reversed fork processing account for the synergistic fork instability. Simultaneous TIM loss and ATR inactivation triggers DNA-PK-dependent CHK1 activation, which is unexpectedly necessary for promoting fork breakage by MRE11 and catastrophic cell death. We propose that acute replisome dysfunction results in a hyper-dependency on ATR to activate local and global fork stabilization mechanisms to counteract irreversible fork collapse. Our study identifies TIM as a point of replication vulnerability in cancer that can be exploited with ATR inhibitors.

The 9-1-1 DNA clamp subunit RAD1 forms specific interactions with clamp loader RAD17, revealing functional implications for binding-protein RHINO

The RAD9-RAD1-HUS1 complex (9-1-1) is a eukaryotic DNA clamp with a crucial role at checkpoints for DNA damage. The ring-like structure of 9-1-1 is opened for loading onto 5' recessed DNA by the clamp loader RAD17 RFC-like complex (RAD17-RLC), in which the RAD17 subunit is responsible for specificity to 9-1-1. Loading of 9-1-1 is required for activation of the ATR-CHK1 checkpoint pathway and the activation is stimulated by a 9-1-1 interacting protein, RHINO, which interacts with 9-1-1 via a recently identified RAD1-binding motif. This discovery led to the hypothesis that other interacting proteins may contain a RAD1-binding motif as well. Here, we show that vertebrate RAD17 proteins also have a putative RAD1-binding motif in their N-terminal regions, and we report the crystal structure of human 9-1-1 bound to a human RAD17 peptide incorporating the motif at 2.1Å resolution. Our structure confirms that the N-terminal region of RAD17 binds to the RAD1 subunit of 9-1-1 via specific interactions. Furthermore, we show that the RAD1-binding motif of RHINO disturbs the interaction of the N-terminal region of RAD17 with 9-1-1. Our results provide deeper understanding of how RAD17-RLC specifically recognizes 9-1-1 and imply that RHINO has a functional role in 9-1-1 loading/unloading and checkpoint activation.

ASPM promotes ATR-CHK1 activation and stabilizes stalled replication forks in response to replication stress.

ASPM is a protein encoded by primary microcephaly 5 (MCPH5) and is responsible for ensuring spindle position during mitosis and the symmetrical division of neural stem cells. We recently reported that ASPM promotes homologous recombination (HR) repair of DNA double strand breaks. However, its potential role in DNA replication and replication stress response remains elusive. Interestingly, we found that ASPM is dispensable for DNA replication under unperturbed conditions. However, ASPM is enriched at stalled replication forks in a RAD17-dependent manner in response to replication stress and promotes RAD9 and TopBP1 loading onto chromatin, facilitating ATR-CHK1 activation. ASPM depletion results in failed fork restart and nuclease MRE11-mediated nascent DNA degradation at the stalled replication fork. The overall consequence is chromosome instability and the sensitization of cancer cells to replication stressors. These data support a role for ASPM in loading RAD17-RAD9/TopBP1 onto chromatin to activate the ATR-CHK1 checkpoint and ultimately ensure genome stability.

Genom-wide analysis identifies single nucleotide polymorphism variations and altered pathways associated with poor semen quality in breeding bulls.

The reason for poor semen quality among the breeding bulls is not well understood. In the present study, we performed high-throughput RNAseq analysis of spermatozoa to identify the SNPs present in good and poor-quality semen-producing Holstein Friesian breeding bulls. A total of 21,360 and 44,650 SNPs were identified in good and poor-quality semen with a minimum read depth of 20, among which 4780 and 8710 novel variants were observed in good and poor-quality semen, respectively. Greater SNPs and indels variations were observed in poor compared to good-quality semen. In poor-quality semen, SNP variations were observed in ZNF280B, SLC26A2, DMXL1, OR52A1, MACROD2 and REV1 genes, which are associated with regulation of spermatogenesis, post-testicular maturation, Cl- channel activity, V-ATPase-mediated intracellular vesicle acidification, a mono-ADP-ribosyl hydrolase and ATR-Chk1 checkpoint activation. GO analysis of filtered genes with significant variations between good and poor-quality semen showed enrichment in important pathways related to semen quality such as MAPK signalling pathway, Akt signalling pathway, focal adhesion, cAMP signalling pathway, and Rap1 signalling pathway. Network analysis of filtered genes in poor-quality semen showed variations in pathways of purine metabolism, pyrimidine metabolism, prolactin signalling pathway and RNA cap-binding complex. It is inferred that SNP in genes involved in maintaining sperm functions could be the reason for poor-quality semen production in bulls, and the identified SNPs hold potential to be used as biomarkers for semen quality in bulls.

Abstract 3722: Extracellular arginine starvation imposes DNA replication fork stall and permits DNA damage tolerance

Abstract Regional nutrients deprivation, which is frequently encountered in many solid tumors due to poor vascularization, induces metabolic rewiring and genetic dysregulation to support the demands of proliferating cancer cells. Numerous chemotherapies exert their S-phase dependent cytotoxicity by producing deleterious effects on DNA to kill proliferating tumor cells. How regional nutrients deprivation affects the response of cancer cells to chemotherapy, especially DNA damaging genotoxicity, and what is the key nutrient dominates these effects is an active area of interest across tumor biology. Of all the amino acids we examined, only arginine is regional deprived in tumor core and indispensable for S-phase progression in multiple breast cancer cells and non-tumorigenic breast epithelial cells. We found that arginine deprivation stalls DNA replication in an mTORC1 inactivation, insufficient metabolites, or the integrated stress response pathway independent manner. We also found that low levels of newly synthesized Histone H4 marks (H4K5ac and H4K12ac), which were reported to be associated with adverse outcomes in various cancers including basal-like and HER2-positive breast cancer, are positively correlated with arginine bioavailability and could serve as surrogate markers of arginine sufficiency. Mechanistically, extracellular arginine is required for the translation of DNA-synthesis-dependent histone H4, but not H3, to promote DNA replication. Knockdown of arginyl-tRNA-transferase, like arginine deprivation, recapitulates the loss of histone H4 lysine 5 and 12 acetylation marks and compromises replication fork speed. Notably, one round of arginine deprivation and addition did not cause an increase in γH2AX, a hallmark of nuclear DNA double-stand breaks. However, multiple rounds of arginine fluctuation promotes the emergence of ATR-Chk1 checkpoint independent-nuclear p53-binding protein 1 bodies in the subsequent G1-phase of daughter cells and induces DNA damage tolerance and resistance to genotoxic agents. Thus, intratumoral variation in the arginine microenvironment synchronizes DNA replication with therapeutic response. Citation Format: Yi-Chang Wang, Andrew A. Kelso, Yi-Hsuan Chen, Chi-An Hsieh, Wei-Kai Chen, Jeremy M. Stark, Hsing-Jien Kung, David K. Ann. Extracellular arginine starvation imposes DNA replication fork stall and permits DNA damage tolerance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3722.

RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling.

RNF8 plays a critical role in DNA damage response (DDR) to initiate ubiquitination-dependent signaling. To better characterize the role of RNF8 in UV-induced DDR, we searched for novel substrates of RNF8 and identified NONO as one intriguing substrate. We found that: (i) RNF8 ubiquitinates NONO and (ii) UV radiation triggers NONO ubiquitination and its subsequent degradation. Depletion of RNF8 inhibited UV-induced degradation of NONO, suggesting that RNF8 targets NONO for degradation in response to UV damage. In addition, we found that 3 NONO lysine residues (positions 279, 290 and 295) are important for conferring its instability in UV-DDR. Depletion of RNF8 or expression of NONO with lysine to arginine substitutions at positions 279, 290 and 295 prolonged CHK1 phosphorylation over an extended period of time. Furthermore, expression of the stable mutant, but not wild-type NONO, induced a prolonged S phase following UV exposure. Stable cell lines expressing the stable NONO mutant showed increased UV sensitivity in a clonogenic survival assay. Since RNF8 recruitment to the UV-damaged sites is dependent on ATR, we propose that RNF8-mediated NONO degradation and subsequent inhibition of NONO-dependent chromatin loading of TOPBP1, a key activator of ATR, function as a negative feedback loop critical for turning off ATR-CHK1 checkpoint signaling in UV-DDR.

SAMHD1 acts at stalled replication forks to prevent interferon induction.

SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.

And-1 coordinates with CtIP for efficient homologous recombination and DNA damage checkpoint maintenance.

To prevent genomic instability, cells respond to DNA lesions by blocking cell cycle progression and initiating DNA repair. Homologous recombination repair of DNA breaks requires CtIP-dependent resection of the DNA ends, which is thought to play a key role in activation of CHK1 kinase to induce the cell cycle checkpoint. But the mechanism is still not fully understood. Here, we establish that And-1, a replisome component, promotes DNA-end resection and DNA repair by homologous recombination. Mechanistically, And-1 interacts with CtIP and regulates CtIP recruitment to DNA damage sites. And-1 localizes to sites of DNA damage dependent on MDC1-RNF8 pathway, and is required for resistance to many DNA-damaging and replication stress-inducing agents. Furthermore, we show that And-1-CtIP axis is critically required for sustained ATR–CHK1 checkpoint signaling and for maintaining both the intra-S- and G2-phase checkpoints. Our findings thus identify And-1 as a novel DNA repair regulator and reveal how the replisome regulates the DNA damage induced checkpoint and genomic stability.

Abstract IA07: Targeting checkpoint signaling and DNA repair in ovarian cancer.

Abstract DNA damage and replication stress activate the ATR-Chk1 checkpoint signaling pathway in which ATR phosphorylates multiple substrates including the kinase Chk1. The ATR-Chk1 signaling pathway regulates DNA repair and cell cycle arrest and promotes the survival of tumor cells exposed to genotoxic chemotherapies. Accordingly, the ATR-Chk1 pathway has emerged as an attractive therapeutic target to increase the antitumor activities of chemotherapy agents, and selective ATR and Chk1 inhibitors have been developed. However, it remains unclear whether ATR and Chk1 inhibition are equally effective with genotoxic chemotherapies that have different mechanisms of action. Here, we compared ATR versus Chk1 inhibition in ovarian cancer cells exposed to agents (cisplatin, topotecan, gemcitabine, and PARP inhibitor) with different mechanisms of action that are used to treat this disease. We found that Chk1 inhibition selectively sensitized to gemcitabine, whereas ATR inhibition broadly sensitized to all chemotherapy agents. Importantly, ATR inhibition also robustly sensitized to PARP inhibitors even in cells with defective HR. These findings suggest that combining ATR inhibitors with these chemotherapy agents may a viable therapeutic strategy in HR-proficient and-deficient tumors. Citation Format: Larry M. Karnitz. Targeting checkpoint signaling and DNA repair in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr IA07.

Abstract C189: Defective S phase cell cycle checkpoint: A potential culprit and target in melanoma

Abstract The S phase cell cycle checkpoint is critical for maintaining genomic integrity. This checkpoint is triggered in response to DNA damage and replication stresses to control cell cycle progression, halting DNA replication and allowing the time to repair to maintain genomic integrity. If this checkpoint is defective, cells lose their ability to ensure the fidelity of replication and repair any damage, resulting in replicative stress and genomic instability which will subsequently increase the risk of cancer. We have discovered that a large proportion of melanoma cell lines lose the ability to halt replication when challenged with high dose hydroxyurea-induced replicative stress, despite normal cell cycle checkpoint signalling. We found that failure in the crosstalk between ATR-CHK1 checkpoint signalling and the cell cycle mechanism in destabilization of CDC25A. Although CHK1-dependent phosphorylation of CDC25A which triggers its degradation was detected, the S phase checkpoint defective melanoma cell lines fail to destabilize CDC25A, maintaining normal activation of CDK2/Cyclin E. The melanoma cells with this S phase checkpoint defect are also hyper-sensitive to killing by CHK1 inhibitor. We have previously shown that melanoma with high levels of replicative stress are very sensitive to CHK1 inhibitors. I will report our studies investigating whether CHK1 inhibitor sensitivity is a direct outcome of the replicative stress arising from the S phase checkpoint defect, or whether the loss of the ATR-CHK1-CDC25A dependent S phase checkpoint signalling mechanism is directly responsible for the hyper-sensitivity to CHK1 inhibitors. Citation Format: Zay Y. Oo, Sheena Daignault, James Chen, Brian Gabrielli. Defective S phase cell cycle checkpoint: A potential culprit and target in melanoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C189.

REV1 is important for the ATR-Chk1 DNA damage response pathway in Xenopus egg extracts

The translesion DNA synthesis (TLS) polymerase REV1 is implicated in the bypass of the irreparable DNA damage such as interstrand crosslinks (ICLs). However, the potential role of REV1 in DNA damage response (DDR) pathway has not been determined. In this research communication, we provide evidence to demonstrate that REV1 plays a previously unidentified but important role in the ATR-Chk1 checkpoint activation in response to mitomycin C (MMC)-induced ICLs in Xenopus egg extracts. We further pinpointed that REV1 plays a downstream role of a checkpoint protein complex assembly including ATR, ATRIP, TopBP1 and the Rad9-Rad1-Hus1 complex to MMC-induced ICLs on chromatin in the DDR pathway. Notably, domain dissection analysis demonstrates that a C-terminal domain, but not the individual ubiquitin binding motifs, of REV1 is important for the binding of REV1 to MMC-damaged chromatin and the MMC-induced Chk1 phosphorylation. Yet, the ATR-Chk1 DDR pathway appears to be dispensable for the preferential association of REV1 to MMC-damaged chromatin. Taken together, REV1 is important for the DDR pathway in Xenopus egg extracts.

Importin β-dependent nuclear import of TopBP1 in ATR–Chk1 checkpoint in Xenopus egg extracts

TopBP1, a multiple-BRCT-containing protein, plays diverse functions in DNA metabolism including DNA replication, DNA damage response and transcriptional regulation. The cytoplasmic localization of TopBP1 has been found to be associated with breast cancer susceptibility in clinical studies, suggesting the biological significance of TopBP1's sub-cellular localization. However, it remains elusive how TopBP1 is shuttled into nucleus and recruited to chromatin under normal or stressful conditions. Taking advantage of Xenopus egg extract, we identified Importin β as a new interacting protein of the TopBP1 C-terminus. We verified the TopBP1–Importin β association via GST pulldown and coimmunoprecipitation assays. We then demonstrated that TopBP1's C-terminal motif (designated as CTM, 23 amino acids) containing a putative NLS (nuclear localization signal) was required for Importin β interaction and that CT100 of Importin β (100 amino acids of extreme C-terminus of Importin β) was required for TopBP1 interaction. Further structure–function analysis reveals that the CTM of TopBP1 is essential for TopBP1's nuclear import and subsequent chromatin recruitment, thereby playing important roles in DNA replication and mitomycin C (MMC)-induced Chk1 phosphorylation. In addition, Importin β-specific inhibitor importazole inhibits TopBP1's nuclear import and the MMC-induced Chk1 phosphorylation. With ongoing DNA replication, the Importin β-dependent nuclear import of TopBP1 was indeed required for the MMC-induced Chk1 phosphorylation. Our data also suggest that checkpoint activation requires more TopBP1 than DNA replication does. The requirement of TopBP1's CTM motif for ATR–Chk1 checkpoint can be bypassed in a nucleus-free AT70 system. Taken together, our findings suggest that the CTM motif-mediated TopBP1 shuttling into nucleus via Importin β plays an important role in the ATR–Chk1 checkpoint signaling in Xenopus egg extracts.

ATR Inhibition Broadly Sensitizes Ovarian Cancer Cells to Chemotherapy Independent of BRCA Status

Replication stress and DNA damage activate the ATR-Chk1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and Chk1 kinases in ovarian cancer cells using genetic and pharmacologic inhibitors in combination with cisplatin, topotecan, gemcitabine, and the PARP inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNA interference (RNAi)-mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan, and gemcitabine each activated Chk1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 nor the Chk1 inhibitor MK-8776 blocked ATR-mediated Chk1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-Chk1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. In addition, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan, and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and Chk1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents and that Chk1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-Chk1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells.

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H2O2) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H2O2-induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H2O2-damaged chromatin. APE2 3'-phosphodiesterase and 3'-5' exonuclease activity is essential for single-stranded DNA generation in the 3'-5' direction from single-stranded breaks, referred to as single-stranded break end resection. In addition, APE2 associates with Chk1, and a serine residue (S86) in the Chk1-binding motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Our data indicate that APE2 plays a vital and previously unexpected role in ATR-Chk1 checkpoint signaling in response to oxidative stress. Thus, our findings shed light on a distinct mechanism of how an ATR-Chk1-dependent DNA damage checkpoint is mediated by APE2 in the oxidative stress response.

UV-induced G2 checkpoint depends on p38 MAPK and minimal activation of ATR-Chk1 pathway

In response to UV light, single-stranded DNA intermediates coated with replication protein A (RPA) are generated, which trigger the ATR-Chk1 checkpoint pathway. Recruitment and/or activation of several checkpoint proteins at the damaged sites is important for the subsequent cell cycle arrest. Surprisingly, upon UV irradiation, Rad9 and RPA only minimally accumulate at DNA lesions in G2 phase, suggesting that only a few single-stranded DNA intermediates are generated. Also, little phosphorylated Chk1 is observed in G2 phase after UV-irradiation, and UV light fails to elicit efficient accumulation of typical DNA damage response proteins at sites of damage in this phase. By contrast, p38 MAPK is phosphorylated in G2 phase cells after UV damage. Interestingly, despite the lack of an obvious activation of the ATR-Chk1 pathway, only the combined inhibition of the ATR- and p38-dependent pathways results in a complete abrogation of the UV-induced G2/M arrest. This suggests that UV light induces less hazardous lesions in G2 phase or that lesions created in this phase are less efficiently processed, resulting in a low activation of the ATR-Chk1 pathway. UV-induced G2 checkpoint activation in this situation therefore relies on signalling via the p38 MAPK and ATR-Chk1 signalling cascades.

Two serine phosphorylation sites in the C‐terminus of Rad9 are critical for 9‐1‐1 binding to TopBP1 and activation of the DNA damage checkpoint response in HeLa cells

A heteromeric proliferating cell nuclear antigen-like ring complex 9-1-1 is comprised of Rad9, Hus1 and Rad1. When assembled, 9-1-1 binds to TopBP1 and activates the ATR-Chk1 checkpoint pathway. This binding in vitro depends on the phosphorylation of Ser-341 and Ser-387 in Rad9 and is reduced to 70% and 20% by an alanine substitution for Ser-341 (S341A) and Ser-387 (S387A), respectively, and to background level by their simultaneous substitution (2A). Here, we show the importance of phosphorylation of these two serine residues in vivo. siRNA-mediated knockdown of Rad9 in HeLa cells impaired UV-induced phosphorylation of checkpoint kinase, Chk1, and conferred hypersensitivity to UV irradiation and to methyl methane sulfonate or hydroxyurea treatments. Either siRNA-resistant wild-type Rad9 (Rad9R(r)) or Rad9R(r) harboring the S341A substitution restored the phosphorylation of Chk1 and damage sensitivity, whereas Rad9R(r) harboring S387A or 2A did not. However, high expression of S387A restored Chk1 phosphorylation and partially suppressed the hypersensitivity. Thus, the affinity of Rad9 to TopBP1 correlates with the activation of the cellular DNA damage response and survival after DNA damage in HeLa cells, and phosphorylation of Ser-341 and Ser-387 of Rad9 is critical for full activation of the checkpoint response to DNA damage.

Abstract 2535: Regulation of Chk1 by the mTOR pathway is essential for cancer cells to complete DNA replication in response to replication stress

Abstract Oncogenic signaling leads to DNA replication stress, DNA damage and consequently DNA damage response (DDR), a barrier for tumorigenesis. For survival and proliferation, cancer cells must overcome this oncogene-induced DDR, but the molecular mechanism by which this is achieved is largely unknown. Here we report that the mTOR pathway prevents either spontaneous or drug induced DNA damage by sustaining ATR-Chk1 checkpoint in pediatric rhabdomyosarcoma (RMS) both in vivo and in vitro. Pharmacological inhibition of mTOR signaling by the selective TOR kinase inhibitor AZD8055 lead to spontaneous DNA damage and apoptosis as demonstrated by the induction of H2AX phosphorylation and PARP1 cleavage both in cell culture and tumor xenografts of RMS. Treatment RMS Rh30 cells with AZD8055 enhanced hydroxyurea induced H2AX phosphorylation and attenuated ATR-Chk1 checkpoint activation, as shown by decreased levels of phospho-Chk1(S345). We further found that mTOR signaling controls CHK1 expression at the transcriptional level via enhancing the activity of cyclin dependent kinases (CDKs). Consistent with its essential role for cells to survive DNA replication stress, siRNA-mediated knockdown of Chk1, or inhibition of Chk1 function by a Chk1 kinase inhibitor resulted in H2AX phosphorylation, while ectopic increase of Chk1 attenuated the DNA damage induced by mTOR kinase inhibition. Most importantly, the mTOR kinase inhibitor prevented the slow S-phase progression following DNA replication stress, which was accompanied with a decrease of Chk1 protein levels and phospho-Chk1(S345). Our data suggest that the TORC1/TORC2 pathway promotes RMS tumorigenesis by enhancing the cell survival functions of the ATR-Chk1 checkpoint in response to DNA replication stress. Our findings discovered an important unexpected functional linkage between TOR signaling and the ATR-Chk1 checkpoint signaling cascade and may impact targeting these two prominent cancer cell signaling pathways. Supported by CA77776. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2535. doi:1538-7445.AM2012-2535

The conserved C terminus of Claspin interacts with Rad9 and promotes rapid activation of Chk1

Claspin is a key mediator of the ATR-Chk1 checkpoint pathway. In response to DNA damage, Claspin interacts with Rad17 and Chk1 in a phosphorylation-dependent manner, enabling ATR to phosphorylate Chk1 efficiently. Claspin also interacts with Rad9, but how they interact and whether the interaction is functional remains unknown. Unexpectedly, our analysis of two splicing isoforms of Claspin provided an answer to these questions. The Claspin1339 isoform contains an evolutionarily conserved C terminus, but the Claspin1332 isoform does not. Although the transcripts encoding both Claspin isoforms coexist in HCT116 cells, Claspin1339 is the predominant form responsible for Chk1 activation. When expressed in cells depleted of endogenous Claspin, both Claspin1339 and Claspin1332 are able to mediate Chk1 activation. However, the activation of Chk1 is delayed in Claspin1332-expressing cells compared with Claspin1339-expressing cells. Furthermore, only Claspin1339 but not Claspin1332 interacts with Rad9 efficiently. Together, these results suggest that the conserved C terminus of Claspin interacts with Rad9 and ensures timely activation of the ATR-Chk1 pathway.