ATM And Rad3-related Research Articles

Factors Influencing the Central Nervous System (CNS) Distribution of the Ataxia Telangiectasia Mutated and Rad3-Related Inhibitor Elimusertib (BAY1895344): Implications for the Treatment of CNS Tumors.

Glioblastoma (GBM) is a disease of the whole brain, with infiltrative tumor cells protected by an intact blood-brain barrier (BBB). GBM has a poor prognosis despite aggressive treatment, in part due to the lack of adequate drug permeability at the BBB. Standard of care GBM therapies include radiation and cytotoxic chemotherapy that lead to DNA damage. Subsequent activation of DNA damage response (DDR) pathways can induce resistance. Various DDR inhibitors, targeting the key regulators of these pathways such as ataxia telangiectasia mutated and Rad3-related (ATR), are being explored as radio- and chemosensitizers. Elimusertib, a novel ATR kinase inhibitor, can prevent repair of damaged DNA, increasing efficacy of DNA-damaging cytotoxic therapies. Robust synergy was observed in vitro when elimusertib was combined with the DNA-damaging agent temozolomide; however, we did not observe improvement with this combination in in vivo efficacy studies in GBM orthotopic tumor-bearing mice. This in vitro-in vivo disconnect was explored to understand factors influencing central nervous system (CNS) distribution of elimusertib and reasons for lack of efficacy. We observed that elimusertib is rapidly cleared from systemic circulation in mice and would not maintain adequate exposure in the CNS for efficacious combination therapy with temozolomide. CNS distribution of elimusertib is partially limited by P-glycoprotein efflux at the BBB, and high binding to CNS tissues leads to low levels of pharmacologically active (unbound) drug in the brain. Acknowledging the potential for interspecies differences in pharmacokinetics, these data suggest that clinical translation of elimusertib in combination with temozolomide for treatment of GBM may be limited. SIGNIFICANCE STATEMENT: This study examined the disconnect between the in vitro synergy and in vivo efficacy of elimusertib/temozolomide combination therapy by exploring systemic and central nervous system (CNS) distributional pharmacokinetics. Results indicate that the lack of improvement in in vivo efficacy in glioblastoma (GBM) patient-derived xenograft (PDX) models could be attributed to inadequate exposure of pharmacologically active drug concentrations in the CNS. These observations can guide further exploration of elimusertib for the treatment of GBM or other CNS tumors.

Molecular basis of CX-5461-induced DNA damage response in primary vascular smooth muscle cells

Our previous studies have shown that the novel selective RNA polymerase I inhibitor CX-5461 suppresses proliferation of vascular smooth muscle cells, mainly by inducing DNA damage response (DDR), including activations of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) and p53. Currently, there is no information about the molecular mechanism(s) underlying CX-5461-induced DDR in vascular cells, while the results obtained in cancer cells and immortalized cell lines are controversial. In this study, we examined the responses of various DDR pathways to CX-5461 treatment in primary aortic smooth muscle cells isolated from normal adult Sprague Dawley rats. We demonstrated that CX-5461-induced DDR was not associated with activations of the nucleotide excision repair, DNA mismatch repair, or the non-homologous end joining pathways, while the homologous recombination pathway was activated. However, the alkaline comet assay did not show massive DNA double strand breaks in CX-5461-treated cells. Instead, CX-5461-induced DDR appeared to be related to induction of DNA replication stress, which was not attributable to increased formation of G-quadruplex or R-loop structures, but might be explained by the increased replication-transcription conflict. CX-5461-induced DDR was not exclusively confined to rDNA within the nucleolar compartment; the extra-nucleolar DDR might represent a distinct secondary response related to the downregulated Rad51 expression in CX-5461-treated cells. In summary, we suggest that DNA replication stress may be the primary molecular event leading to downstream ATM/ATR and p53 activations in CX-5461-treated vascular smooth muscle cells. Our results provide further insights into the molecular basis of the beneficial effects of CX-5461 in proliferative vascular diseases.

Pharmacodynamic (PD) and immunophenotyping analyses of ATR inhibitor (ATRi) tuvusertib + ATM inhibitor (ATMi) lartesertib in a phase Ib study in patients with advanced unresectable solid tumors.

2612 Background: Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) protein kinases orchestrate the DNA damage response. A synthetic lethal relationship between ATR and ATM genes in cancer has been described1 and ATMi synergistically potentiate the efficacy of ATRi in vitro and in vivo.2 The combination of tuvusertib + lartesertib was investigated in Part A1 of the open-label, multicenter study DDRiver Solid Tumors 320 in patients with advanced unresectable solid tumors. Here, we report results of the PD and immunophenotyping analyses. Methods: PD analysis comprised γ-H2AX in serial blood samples, stimulated ex vivowith 4-NQO, bleomycin, or controls. Flow cytometry was used to measure target inhibition via γ-H2AX modulation in the CD45+ lymphocytes fraction and to explore the effect of tuvusertib + lartesertib on the immunophenotype (myeloid-derived suppressor cells, T and B lymphocytes, monocytes, and natural killer cells subsets) in serial blood samples. Pharmacokinetic samples were analyzed by a validated bioanalytical liquid chromatography/mass spectrometry method. Time-matched blood samples were collected at baseline, 1, 3, 6, and 24 hours after first tuvusertib and lartesertib administration on days 1 and 8 of cycle 1 for the γ-H2AX analysis, and on days 1 and 15 of cycles 1 and 2 before treatment for immunophenotyping. Results: Immunophenotyping data and γ-H2AX levels were obtained from 41 and 34 patients, respectively. For tuvusertib, complete or almost complete target inhibition was seen at 1–6 hours after treatment, followed by a rebound above baseline after 24 hours, on both days 1 and 8 at doses of 130 and 180 mg once daily (QD). For lartesertib, a variable target inhibition of approximately 50 % on average was seen across all time points at doses of 100, 150 and 200 mg QD. No target inhibition was seen for tuvusertib at 90 mg QD and lartesertib at 50 mg QD (cohort 1). Tuvusertib + lartesertib induced a transient decrease of monocytes and natural killer (NK) cells, with partial or complete recovery to baseline levels during treatment breaks in schedules of 2 weeks on treatment followed by a treatment break of 1 or 2 weeks, respectively. Conclusions: Tuvusertib and lartesertib combination PD outcomes were in line with respective monotherapy observations.3,4 The combination did not cause any consistent change in the levels of immune cell subsets at all dose levels tested, except a mild, transient decrease in monocytes and NK cells, in line with the tuvusertib monotherapy observations. 1. Kantidze et al., Trends Cancer 2018;4(11):755–68. 2. Turchick et al., Mol Cancer Ther 2023;22(7):859–72. 3. Yap et al., Ann Oncol 2022;33(suppl_7):S197–S224. 4. Siu et al., Cancer Res 2023;83(8_Suppl):CT171. Clinical trial information: NCT05396833 .

Insights into the Dissociation Process and Binding Pattern of the BRCT7/8-PHF8 Complex.

DNA topoisomerase 2-binding protein 1 (Topbp1) plays a crucial role in activating the ataxia-telangiectasia mutated and rad3-related (ATR) complex to initiate DNA damage repair responses. For this process to occur, it is necessary for PHF8 to dissociate from Topbp1. Topbp1 binds to the acidic patch sequence (APS) of PHF8 through its C-terminal BRCT7/8 domain, and disrupting this interaction could be a promising strategy for cancer treatment. To investigate the dissociation process and binding pattern of BRCT7/8-PHF8, we employed enhanced sampling techniques, such as steered molecular dynamics (SMD) simulations and accelerated molecular dynamics (aMD) simulations, along with self-organizing maps (SOM) and time-resolved force distribution analysis (TRFDA) methodologies. Our results demonstrate that the dissociation of PHF8 from BRCT7/8 starts from the N-terminus, leading to the unfolding of the N-terminal helix. Additionally, we identified critical residues that play a pivotal role in this dissociation process. These findings provide valuable insights into the disassociation of PHF8 from BRCT7/8, which could potentially guide the development of novel drugs targeting Topbp1 for cancer therapy.

Lighting ATR/Chk1 by mesoscale TopBP1 condensates

Biomolecular condensation has gained considerable attention as a fundamental mechanism in cell signaling and various biological processes. A recent study by Egger et al. provides valuable insights into the constituents of topoisomerase IIβ binding protein 1 (TopBP1) condensates and sheds light on the mechanism of Chk1 activation by ataxia telangiectasia-mutated and Rad3-related (ATR) at the interface of these condensates.

Spatial organization and functions of Chk1 activation by TopBP1 biomolecular condensates

Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1's ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway.

ATR limits Rad18-mediated PCNA monoubiquitination to preserve replication fork and telomerase-independent telomere stability.

Upon replication fork stalling, the RPA-coated single-stranded DNA (ssDNA) formed behind the fork activates the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase, concomitantly initiating Rad18-dependent monoubiquitination of PCNA. However, whether crosstalk exists between these two events and the underlying physiological implications of this interplay remain elusive. In this study, we demonstrate that during replication stress, ATR phosphorylates human Rad18 at Ser403, an adjacent residue to a previously unidentified PIP motif (PCNA-interacting peptide) within Rad18. This phosphorylation event disrupts the interaction between Rad18 and PCNA, thereby restricting the extent of Rad18-mediated PCNA monoubiquitination. Consequently, excessive accumulation of the tumor suppressor protein SLX4, now characterized as a novel reader of ubiquitinated PCNA, at stalled forks is prevented, contributing to the prevention of stalled fork collapse. We further establish that ATR preserves telomere stability in alternative lengthening of telomere (ALT) cells by restricting Rad18-mediated PCNA monoubiquitination and excessive SLX4 accumulation at telomeres. These findings shed light on the complex interplay between ATR activation, Rad18-dependent PCNA monoubiquitination, and SLX4-associated stalled fork processing, emphasizing the critical role of ATR in preserving replication fork stability and facilitating telomerase-independent telomere maintenance.

LncRNA HOTAIR promotes DNA damage repair and radioresistance by targeting ATR in colorectal cancer.

Long non-coding RNAs (lncRNAs) have been implicated in cancer progression and drug resistance development. Moreover, there is evidence that lncRNA HOX transcript antisense intergenic RNA (HOTAIR) is involved in colorectal cancer (CRC) progression. The present study aimed to examine the functional role of lncRNA HOTAIR in conferring radiotherapy resistance in CRC cells, as well as the underlying mechanism. The relative expression levels of HOTAIR were examined in 70 pairs of CRC tumor and para-cancerous tissues, as well as in radiosensitive and radioresistant samples. The correlations between HOTAIR expression levels and clinical features of patients with CRC were assessed using the Chi-square test. Functional assays such as cell proliferation, colony formation and apoptosis assays were conducted to determine the radiosensitivity in CRC cells with HOTAIR silencing after treatment with different doses of radiation. RNA pull-down assay and fluorescence in situ hybridization (FISH) were used to determine the interaction between HOTAIR and DNA damage response mediator ataxia-telangiectasia mutated- and Rad3-related (ATR). HOTAIR was significantly upregulated in CRC tumor tissues, especially in radioresistant tumor samples. The elevated expression of HOTAIR was correlated with more advanced histological grades, distance metastasis and the poor prognosis in patients with CRC. Silencing HOTAIR suppressed the proliferation and promoted apoptosis and radiosensitivity in CRC cells. HOTAIR knockdown also inhibited the tumorigenesis of CRC cells and enhanced the sensitivity to radiotherapy in a mouse xenograft model. Moreover, the data showed that HOTAIR could interact with ATR to regulate the DNA damage repair signaling pathway. Silencing HOTAIR impaired the ATR-ATR interacting protein (ATRIP) complex and signaling in cell cycle progression. Collectively, the present results indicate that lncRNA HOTAIR facilitates the DNA damage response pathway and promotes radioresistance in CRC cells by targeting ATR.

Abstract LB_A10: Efficacy of ATR inhibitor berzosertib plus topotecan or topotecan alone in patients with relapsed small cell lung cancer: A randomized clinical trial

Abstract Background: Patients with relapsed small cell lung cancer (SCLC) have a poor prognosis and few therapeutic options, especially after standard treatment of platinum doublet chemotherapy +/- immunotherapy. SCLC is characterized with high replication stress. A previous single-arm phase 2 study showed anti-tumor activity with the addition of ataxia-telangiectasia–mutated and rad3-related (ATR) kinase inhibitor berzosertib to topotecan. This study aimed to investigate whether the addition of berzosertib to topotecan improves clinical outcomes in patients with relapsed SCLC. Patients and methods: The randomized, open-labeled phase 2 study recruited patients with SCLC and relapse after one or more prior therapies from 16 US cancer centers as members of the National Cancer Institute Cancer Therapy Evaluation Program. Patients previously treated with topotecan were not eligible. Eligible patients were randomly assigned to receive topotecan (arm 1: 1.25 mg/m2 intravenously on days 1 through 5) alone or with berzosertib (arm 2: 210 mg/m2 intravenously on days 2 and 5), in 21-day cycles. Randomization was stratified by sensitivity to first-line platinum-based chemotherapy. The primary endpoint was progression-free survival (PFS) in the intention-to-treat population. Secondary endpoints included overall survival (OS), objective response rate (ORR), and duration of response (DOR) in the overall population and in platinum-sensitive or -resistant patients. Results: Between December 2019 and December 2022, sixty patients were randomly assigned to receive topotecan alone (n=20) or in combination of with berzosertib (n=40). After a median potential follow-up of 21.3 months, there was no difference in PFS between the two arms [median: 3.0 months in arm 1 versus 3.9 months in arm 2, hazard ratio (HR) (95% CI): 0.80 (0.46 to 1.41), P = 0.44]. OS was significantly longer with the combination [5.4 months in arm 1 versus 8.9 months in arm 2, HR (95% CI): 0.53 (0.29 to 0.96), P = 0.033]. Of 57 evaluable patients, a numerically higher ORR was observed in patients receiving the combination: 5.6% (1/18, 95% CI: 0.1% to 27.3%) in arm 1 versus 25.6% (10/39, 13.0% to 42.1%) in arm 2. In an unplanned post hoc analysis, the depth of target lesion responses was significantly associated with better OS in patients on the combination arm (adjusted P = 0.022 by landmark analysis), suggesting that the deeper tumor regressions by the combination of berzosertib and topotecan may have eventually contributed to OS improvement. Adverse event profiles were similar between the two arms. Conclusions: In this randomized clinical trial, berzosertib plus topotecan did not improve PFS compared with topotecan alone in patients with relapsed SCLC. However, the combination significantly improved OS, suggesting potential efficacy of replication-stress targeted therapies in SCLC. Citation Format: Nobuyuki Takahashi, Zhonglin Hao, Liza C Villaruz, Jun Zhang, Jimmy Ruiz, W. Jeffrey Petty, Hirva Mamdani, Jonathan W Riess, Jorge Nieva, Jose M Pachecho, Alexander D Fuld, Elaine Shum, Aman Chauhan, Samantha Nichols, Hirity Shimellis, Jessie McGlone, Linda Sciuto, Danielle Pinkiert, Chante Graham, Meenakshi Shelat, Robbie Kattappuram, Melissa Abel, Brett Schroeder, Deep Upadhyay, Manan Krishnamurthy, Ajit Kumar Sharma, Rajesh Kumar, Justin Malin, Christopher W Schultz, Shubhank Goyal, Christophe E Redon, Yves Pommier, Mirit I Aladjem, Steven D Gore, Seth M Steinberg, Rasa Vilimas, Parth Desai, Anish Thomas. Efficacy of ATR inhibitor berzosertib plus topotecan or topotecan alone in patients with relapsed small cell lung cancer: A randomized clinical trial [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A10.

Targeting TP53 disruption in chronic lymphocytic leukemia: Current strategies and future directions.

In the modern era of Chronic Lymphocytic Leukemia (CLL) targeted therapy, the loss of p53 function due to genetic abnormalities remains a significant challenge. This is because even targeted agents, which are currently the mainstay of treatment for CLL, do not directly target p53 or restore its disrupted pathway. Consequently, resistance to therapy and unfavorable clinical outcomes often accompany these p53-related abnormalities. An essential goal of future clinical research should be to address the ostensibly "undruggable" p53 pathway. Currently, multiple therapeutic approaches are being explored to tackle TP53 dysfunction and improve outcomes in high-risk CLL. These approaches include the use of oncoprotein murine double minute 2 inhibitors, small-molecule p53 reactivators, exportin 1 (XPO1) inhibitors, and ataxia-telangiectasia mutated and Rad3-related (ATR) inhibitors. Combinations of these p53-targeting strategies, along with established novel therapies such as B-cell receptor or B-cell lymphoma-2 (BCL-2) inhibitors, may shape the future of therapeutic trials in this challenging-to-treat disease.

RASSF1A promotes ATM signaling and RASSF1A methylation is a synthetic lethal marker for ATR inhibitors.

Aim: The mechanism of RASSF1A in DNA damage repair remains to be further clarified for applying to synthetic lethal strategy. Materials & methods: Eight esophageal cancer cell lines, 181 cases of esophageal dysplasia and 1066 cases of primary esophageal squamous cell carcinoma (ESCC) were employed. Methylation-specific PCR, theCRISPR/Cas9 technique, immunoprecipitation assay and a xenograft mouse model were used. Results: RASSF1A was methylated in 2.21% of esophageal dysplasia and 11.73% of ESCC. RASSF1A was alsoinvolved in DNA damage repair through activating Hippo signaling. Loss of RASSF1A expression sensitized esophageal cancer cell lines to ataxia telangiectasia mutated and rad3-related (ATR) inhibitor (VE-822) both in vitro and in vivo. Conclusion: RASSF1A methylation is a synthetic lethal marker for ATR inhibitors.

RP11-789C1.1 inhibits gastric cancer cell proliferation and accelerates apoptosis via the ATR/CHK1 signaling pathway.

Long non-coding RNAs (lncRNAs) plays an important role in the progression of gastric cancer (GC). Their involvement ranges from genetic regulation to cancer progression. However, the mechanistic roles of RP11-789C1.1 in GC are not fully understood. We identified the expression of lncRNA RP11-789C1.1 in GC tissues and cell lines by real-time fluorescent quantitative polymerase chain reaction. A series of functional experiments revealed the effect of RP11-789C1.1 on the proliferation of GC cells. In vivo experiments verified the effect of RP11-789C1.1 on the biological behavior of a GC cell line. RNA pull-down unveiled RP11-789C1.1 interacting proteins. Western blot analysis indicated the downstream pathway changes of RP11-789C1.1, and an oxaliplatin dosing experiment disclosed the influence of RP11-789C1.1 on the drug sensitivity of oxaliplatin. Our results demonstrated that RP11-789C1.1 inhibited the proliferation of GC cells and promoted the apoptosis of GC cells. Mechanistically, RP11-789C1.1 inhibited checkpoint kinase 1 (CHK1) phosphorylation by binding ataxia-telangiectasia mutated and Rad3 related (ATR), a serine/threonine-specific protein kinase, promoted GC apoptosis, and mediated oxaliplatin sensitivity. In general, we discovered a tumor suppressor molecule RP11-789C1.1 and confirmed its mechanism of action, providing a theoretical basis for targeted GC therapy.

ATR promotes clearance of damaged DNA and damaged cells by rupturing micronuclei

The human ataxia telangiectasia mutated and Rad3-related (ATR) kinase functions in the nucleus to protect genomic integrity. Micronuclei (MN) arise from genomic and chromosomal instability and cause aneuploidy and chromothripsis, but how MN are removed is poorly understood. Here, we show that ATR is active in MN and promotes their rupture in S phase by phosphorylating Lamin A/C at Ser395, which primes Ser392 for CDK1 phosphorylation and destabilizes the MN envelope. In cells harboring MN, ATR or CDK1 inhibition reduces MN rupture. Consequently, ATR inhibitor (ATRi) diminishes activation of the cytoplasmic DNA sensor cGAS and compromises cGAS-dependent autophagosome accumulation in MN and clearance of micronuclear DNA. Furthermore, ATRi reduces cGAS-mediated senescence and killing of MN-bearing cancer cells by natural killer cells. Thus, in addition to the canonical ATR signaling pathway, an ATR-CDK1-Lamin A/C axis promotes MN rupture to clear damaged DNA and cells, protecting the genome in cell populations through unexpected cell-autonomous and cell-non-autonomous mechanisms.

MEF2A suppresses stress responses that trigger DDX41-dependent IFN production

Cellular stress in the form of disrupted transcription, loss of organelle integrity, or damage to nucleic acids can elicit inflammatory responses by activating signaling cascades canonically tasked with controlling pathogen infections. These stressors must be kept in check to prevent unscheduled activation of interferon, which contributes to autoinflammation. This study examines the role of the transcription factor myocyte enhancing factor 2A (MEF2A) in setting the threshold of transcriptional stress responses to prevent R-loop accumulation. Increases in R-loops lead to the induction of interferon and inflammatory responses in a DEAD-box helicase 41 (DDX41)-, cyclic GMP-AMP synthase (cGAS)-, and stimulator of interferon genes (STING)-dependent manner. The loss of MEF2A results in the activation of ATM and RAD3-related (ATR) kinase, which is also necessary for the activation of STING. This study identifies the role of MEF2A in sustaining transcriptional homeostasis and highlights the role of ATR in positively regulating R-loop-associated inflammatory responses.

Design, Synthesis, and Biological Evaluation of Potent and Selective Inhibitors of Ataxia Telangiectasia Mutated and Rad3-Related (ATR) Kinase for the Efficient Treatment of Cancer.

Ataxia telangiectasia mutated and Rad3-related (ATR), a vital member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, plays a critical role in the DNA damage response (DDR). Tumor cells with a loss of DDR function or defects in the ataxia telangiectasia mutated (ATM) gene are generally more dependent on ATR for survival, suggesting that ATR is an attractive anticancer drug target based on its synthetic lethality. Herein, we present a potent and highly selective ATR inhibitor, ZH-12 (IC50 = 0.0068 μM). It showed potent antitumor activity as a single agent or in combination with cisplatin in the human colorectal adenocarcinoma LoVo tumor xenograft mouse model. Overall, ZH-12 may be a promising ATR inhibitor based on the principle of synthetic lethality and deserves further in-depth study.

P130RB2 positively contributes to ATR activation in response to replication stress via the RPA32-ETAA1 axis

Ataxia-telangiectasia mutated and Rad3-related (ATR) kinase is a crucial regulator of the cell cycle checkpoint and activated in response to DNA replication stress by two independent pathways via RPA32-ETAA1 and TopBP1. However, the precise activation mechanism of ATR by the RPA32-ETAA1 pathway remains unclear. Here, we show that p130RB2, a member of the retinoblastoma protein family, participates in the pathway under hydroxyurea-induced DNA replication stress. p130RB2 binds to ETAA1, but not TopBP1, and depletion of p130RB2 inhibits the RPA32-ETAA1 interaction under replication stress. Moreover, p130RB2 depletion reduces ATR activation accompanied by phosphorylation of its targets RPA32, Chk1, and ATR itself. It also causes improper re-progression of S phase with retaining single-stranded DNA after cancelation of the stress, which leads to an increase in the anaphase bridge phenotype and a decrease in cell survival. Importantly, restoration of p130RB2 rescued the disrupted phenotypes of p130RB2 knockdown cells. These results suggest positive involvement of p130RB2 in the RPA32-ETAA1-ATR axis and proper re-progression of the cell cycle to maintain genome integrity.

ATM and ATR, two central players of the DNA damage response, are involved in the induction of systemic acquired resistance by extracellular DNA, but not the plant wound response.

The plant immune response to DNA is highly self/nonself-specific. Self-DNA triggered stronger responses by early immune signals such as H2O2 formation than nonself-DNA from closely related plant species. Plants lack known DNA receptors. Therefore, we aimed to investigate whether a differential sensing of self-versus nonself DNA fragments as damage- versus pathogen-associated molecular patterns (DAMPs/PAMPs) or an activation of the DNA-damage response (DDR) represents the more promising framework to understand this phenomenon. We treated Arabidopsis thaliana Col-0 plants with sonicated self-DNA from other individuals of the same ecotype, nonself-DNA from another A. thaliana ecotype, or nonself-DNA from broccoli. We observed a highly self/nonself-DNA-specific induction of H2O2 formation and of jasmonic acid (JA, the hormone controlling the wound response to chewing herbivores) and salicylic acid (SA, the hormone controlling systemic acquired resistance, SAR, to biotrophic pathogens). Mutant lines lacking Ataxia Telangiectasia Mutated (ATM) or ATM AND RAD3-RELATED (ATR) - the two DDR master kinases - retained the differential induction of JA in response to DNA treatments but completely failed to induce H2O2 or SA. Moreover, we observed H2O2 formation in response to in situ-damaged self-DNA from plants that had been treated with bleomycin or SA or infected with virulent bacteria Pseudomonas syringae pv. tomato DC3000 or pv. glycinea carrying effector avrRpt2, but not to DNA from H2O2-treated plants or challenged with non-virulent P. syringae pv. glycinea lacking avrRpt2. We conclude that both ATM and ATR are required for the complete activation of the plant immune response to extracellular DNA whereas an as-yet unknown mechanism allows for the self/nonself-differential activation of the JA-dependent wound response.

The ATR-WEE1 kinase module promotes SUPPRESSOR OF GAMMA RESPONSE 1 translation to activate replication stress responses.

DNA replication stress threatens genome stability and is a hallmark of cancer in humans. The evolutionarily conserved kinases ATR (ATM and RAD3-related) and WEE1 are essential for the activation of replication stress responses. Translational control is an important mechanism that regulates gene expression, but its role in replication stress responses is largely unknown. Here we show that ATR-WEE1 control the translation of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a master transcription factor required for replication stress responses in Arabidopsis thaliana. Through genetic screening, we found that the loss of GENERAL CONTROL NONDEREPRESSIBLE 20 (GCN20) or GCN1, which function together to inhibit protein translation, suppressed the hypersensitivity of the atr or wee1 mutant to replication stress. Biochemically, WEE1 inhibits GCN20 by phosphorylating it; phosphorylated GCN20 is subsequently polyubiquitinated and degraded. Ribosome profiling experiments revealed that that loss of GCN20 enhanced the translation efficiency of SOG1, while overexpressing GCN20 had the opposite effect. The loss of SOG1 reduced the resistance of wee1 gcn20 to replication stress, whereas overexpressing SOG1 enhanced the resistance to atr or wee1 to replication stress. These results suggest that ATR-WEE1 inhibits GCN20-GCN1 activity to promote the translation of SOG1 during replication stress. These findings link translational control to replication stress responses in Arabidopsis.

Discovery of Thieno[3,2-d]pyrimidine derivatives as potent and selective inhibitors of ataxia telangiectasia mutated and Rad3 related (ATR) kinase

The ataxia telangiectasia mutated and rad3-related (ATR) kinase regulates the DNA damage response (DDR), which plays a critical role in the ATR-Chk1 signaling pathway. ATR inhibition can induce synthetic lethality (SL) with several DDR deficiencies, making it an attractive drug target for cancers with DDR defects. In this study, we developed a series of selective and potent ATR inhibitors with a thieno[3,2-d]pyrimidine scaffold using a hybrid design. We identified compound 34 as a representative molecule that inhibited ATR kinase with an IC50 value of 1.5 nM and showed reduced potency against other kinases tested. Compound 34 also exhibited potent antiproliferative effects against LoVo cells and SL effects against HT-29 cells. Moreover, compound 34 demonstrated good pharmacokinetic properties, in vivo antitumor efficacy, and no obvious toxicity in the LoVo xenograft tumor model. Therefore, compound 34 is a promising lead compound for drug development to combat specific DDR deficiencies in cancer patients.

Design, synthesis, and biological evaluation of pyrido[3,2-d]pyrimidine derivatives as novel ATR inhibitors

Targeting ataxia telangiectasia mutated and Rad3-related (ATR) kinase is being pursued as a new therapeutic strategy for the treatment of advanced solid tumor with specific DNA damage response deficiency. Herein, we report a series of pyrido[3,2-d]pyrimidine derivatives with potent ATR inhibitory activity through structure-based drug design. Among them, the representative compound 10q exhibited excellent potency against ATR in both biochemical and cellular assays. More importantly, 10q exhibited good liver microsomes stability in different species and also showed moderate inhibitory activity against HT-29 cells in combination treatment with the ATM inhibitor AZD1390. Thus, this work provides a promising lead compound against ATR for further study.