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Increased Replication Stress Determines ATR Inhibitor Sensitivity in Neuroblastoma Cells.

ABSTRACT: Despite intensive high-dose multimodal therapy, high-risk neuroblastoma (NB) confers a less than 50% survival rate. This study investigates the role of replication stress in sensitivity to inhibition of Ataxia telangiectasia and Rad3-related (ATR) in pre-clinical models of high-risk NB. Amplification of the oncogene MYCN always imparts high-risk disease and occurs in 25% of all NB. Here, we show that MYCN-induced replication stress directly increases sensitivity to the ATR inhibitors VE-821 and AZD6738. PARP inhibition with Olaparib also results in replication stress and ATR activation, and sensitises NB cells to ATR inhibition independently of MYCN status, with synergistic levels of cell death seen in MYCN expressing ATR- and PARP-inhibited cells. Mechanistically, we demonstrate that ATR inhibition increases the number of persistent stalled and collapsed replication forks, exacerbating replication stress. It also abrogates S and G2 cell cycle checkpoints leading to death during mitosis in cells treated with an ATR inhibitor combined with PARP inhibition. In summary, increased replication stress through high MYCN expression, PARP inhibition or chemotherapeutic agents results in sensitivity to ATR inhibition. Our findings provide a mechanistic rationale for the inclusion of ATR and PARP inhibitors as a potential treatment strategy for high-risk NB.

SUBMITTER: King D

PROVIDER: S-EPMC8699051 | biostudies-literature |

REPOSITORIES: biostudies-literature

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MYCN expression induces replication stress and sensitivity to PARP inhibition in neuroblastoma.

Project description:This study investigates the influence expression of the MYCN oncogene has on the DNA damage response, replication fork progression and sensitivity to PARP inhibition in neuroblastoma. In a panel of neuroblastoma cell lines, MYCN amplification or MYCN expression resulted in increased cell death in response to a range of PARP inhibitors (niraparib, veliparib, talazoparib and olaparib) compared to the response seen in non-expressing/amplified cells. MYCN expression slowed replication fork speed and increased replication fork stalling, an effect that was amplified by PARP inhibition or PARP1 depletion. Increased DNA damage seen was specifically induced in S-phase cells. Importantly, PARP inhibition caused a significant increase in the survival of mice bearing MYCN expressing tumours in a transgenic murine model of MYCN expressing neuroblastoma. Olaparib also sensitized MYCN expressing cells to camptothecin- and temozolomide-induced cell death to a greater degree than non-expressing cells. In summary, MYCN expression leads to increased replication stress in neuroblastoma cells. This effect is exaggerated by inhibition of PARP, resulting in S-phase specific DNA damage and ultimately increased tumour cell death. PARP inhibition alone or in combination with classical chemotherapeutics is therefore a potential therapeutic strategy for neuroblastoma and may be more effective in MYCN expressing tumours.

| S-EPMC7289530 | biostudies-literature

Loss of NEIL3 DNA glycosylase markedly increases replication associated double strand breaks and enhances sensitivity to ATR inhibitor in glioblastoma cells.

Project description:DNA endonuclease eight-like glycosylase 3 (NEIL3) is one of the DNA glycosylases that removes oxidized DNA base lesions from single-stranded DNA (ssDNA) and non-B DNA structures. Approximately seven percent of human tumors have an altered NEIL3 gene. However, the role of NEIL3 in replication-associated repair and its impact on modulating treatment response is not known. Here, we report that NEIL3 is localized at the DNA double-strand break (DSB) sites during oxidative DNA damage and replication stress. Loss of NEIL3 significantly increased spontaneous replication-associated DSBs and recruitment of replication protein A (RPA). In contrast, we observed a marked decrease in Rad51 on nascent DNA strands at the replication fork, suggesting that HR-dependent repair is compromised in NEIL3-deficient cells. Interestingly, NEIL3-deficient cells were sensitive to ataxia-telangiectasia and Rad3 related protein (ATR) inhibitor alone or in combination with PARP1 inhibitor. This study elucidates the mechanism by which NEIL3 is critical to overcome oxidative and replication-associated genotoxic stress. Our findings may have important clinical implications to utilize ATR and PARP1 inhibitors to enhance cytotoxicity in tumors that carry altered levels of NEIL3.

| S-EPMC5762564 | biostudies-literature

Sensitivity of cells to ATR and CHK1 inhibitors requires hyperactivation of CDK2 rather than endogenous replication stress or ATM dysfunction.

Project description:DNA damage activates cell cycle checkpoint proteins ATR and CHK1 to arrest cell cycle progression, providing time for repair and recovery. Consequently, inhibitors of ATR (ATRi) and CHK1 (CHK1i) enhance damage-induced cell death. Intriguingly, both CHK1i and ATRi alone elicit cytotoxicity in some cell lines. Sensitivity has been attributed to endogenous replications stress, but many more cell lines are sensitive to ATRi than CHK1i. Endogenous activation of the DNA damage response also did not correlate with drug sensitivity. Sensitivity correlated with the appearance of γH2AX, a marker of DNA damage, but without phosphorylation of mitotic markers, contradicting suggestions that the damage is due to premature mitosis. Sensitivity to ATRi has been associated with ATM mutations, but dysfunction in ATM signaling did not correlate with sensitivity. CHK1i and ATRi circumvent replication stress by reactivating stalled replicons, a process requiring a low threshold activity of CDK2. In contrast, γH2AX induced by single agent ATRi and CHK1i requires a high threshold activity CDK2. Hence, phosphorylation of different CDK2 substrates is required for cytotoxicity induced by replication stress plus ATRi/CHK1i as compared to their single agent activity. In summary, sensitivity to ATRi and CHK1i as single agents is elicited by premature hyper-activation of CDK2.

| S-EPMC8007816 | biostudies-literature

AXL Inhibition Induces DNA Damage and Replication Stress in Non-Small Cell Lung Cancer Cells and Promotes Sensitivity to ATR Inhibitors.

Project description:AXL, a TAM (TYRO3, AXL, and MERTK) family receptor tyrosine kinase, is increasingly being recognized as a key determinant of resistance to targeted therapies, as well as chemotherapy and radiation in non-small cell lung cancer (NSCLC) and other cancers. We further show here that high levels of AXL and epithelial-to-mesenchymal transition were frequently expressed in subsets of both treatment-naïve and treatment-relapsed NSCLC. Previously, we and others have demonstrated a role for AXL in mediating DNA damage response (DDR), as well as resistance to inhibition of WEE1, a replication stress response kinase. Here, we show that BGB324 (bemcentinib), a selective small-molecule AXL inhibitor, caused DNA damage and induced replication stress, indicated by ATR/CHK1 phosphorylation, more significantly in TP53-deficient NSCLC cell lines. Similar effects were also observed in large-cell neuroendocrine carcinoma (LCNEC) cell lines. High AXL protein levels were also associated with resistance to ATR inhibition. Combined inhibition of AXL and ATR significantly decreased cell proliferation of NSCLC and LCNEC cell lines. Mechanistically, combined inhibition of AXL and ATR significantly increased RPA32 hyperphosphorylation and DNA double-strand breaks and induced markers of mitotic catastrophe. Notably, NSCLC cell lines with low levels of SLFN11, a known predictive biomarker for platinum and PARP inhibitor sensitivity, were more sensitive to AXL/ATR cotargeting. These findings demonstrate a novel and unexpected role for AXL in replication stress tolerance, with potential therapeutic implications. IMPLICATIONS: These findings demonstrate that the combination of AXL and ATR inhibitors could be a promising therapeutic combination for NSCLC, LCNEC, and other cancers.

| S-EPMC7925356 | biostudies-literature

Pharmacological degradation of ATR induces antiproliferative DNA replication stress in leukemic cells.

Project description:Mammalian cells replicate ~ 3 × 109 base pairs per cell cycle. One of the key molecules that slows down the cell cycle and prevents excessive DNA damage upon DNA replication stress is the checkpoint kinase ataxia-telangiectasia-and-RAD3-related (ATR). Proteolysis-targeting-chimeras (PROTACs) are an innovative pharmacological invention to molecularly dissect, biologically understand, and therapeutically assess catalytic and non-catalytic functions of enzymes. This work defines the first-in-class ATR PROTAC, Abd110/Ramotac-1. It is derived from the ATR inhibitor VE-821 and recruits the E3 ubiquitin-ligase component cereblon to ATR. Abd110 eliminates ATR rapidly in human leukemic cells. This mechanism provokes DNA replication catastrophe and augments anti-leukemic effects of the clinically used ribonucleotide reductase-2 inhibitor hydroxyurea. Moreover, Abd110 is more effective than VE-821 against human primary leukemic cells but spares normal primary immune cells. CRISPR-Cas9 screens show that ATR is a dependency factor in 116 myeloid and lymphoid leukemia cells. Treatment of wild-type but not of cereblon knockout cells with Abd110 stalls their proliferation which verifies that ATR elimination is the primary mechanism of Abd110. Altogether, our findings demonstrate specific anti-leukemic effects of an ATR PROTAC.

| S-EPMC11306515 | biostudies-literature

Loss of Cyclin C or CDK8 provides ATR inhibitor resistance by suppressing transcription-associated replication stress.

Project description:The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR-Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death.

| S-EPMC8421211 | biostudies-literature

The ATR inhibitor VE-821 increases the sensitivity of gastric cancer cells to cisplatin.

Project description:BackgroundChemoresistance is a common event after cancer chemotherapy, including gastric cancer (GC). Cisplatin has been reported to induce the DNA damage response (DDR), thus leading to chemoresistance. VE-821, a specific inhibitor of ATR, has been proven to suppress a variety of solid malignancies effectively. Our study aimed to explore the effect of VE-821 on enhancing the chemical sensitivity to cisplatin and clarify the potential molecular mechanisms.MethodsCell viability and apoptosis of MKN-45 and AGS were measured by CCK8 and flow cytometry assay respectively. Western blotting was used to detect the expression of target proteins. TCGA database was used to analyze the correlation between the ATR expression with the prognosis of GC patients. The viability of GC organoids was detected by Cell Titer Glo (CTG) through luminescence.ResultsCisplatin inhibited the proliferation and induced apoptosis of GC cells with a relatively high IC50 value, and increased the phosphorylation levels of ATR-CHK1 and H2AX. VE-821 achieved the same effects but by downregulating the phosphorylation levels of the ATR-CHK1 pathway. Besides, higher ATR expression in GC tissues was positively correlated with higher pathological stage in GC patients. Interestingly, ATR inhibition reversed cisplatin-induced STAT3 activation and enhanced H2AX levels. Moreover, VE-821 significantly sensitized GC cells to cisplatin, and these two drugs had synergistic effects in GC cell lines, organoids, and in vivo.ConclusionOur results suggested VE-821 sensitized GC cells to cisplatin via reversing DDR activation. And VE-821 treatment may be a promising therapeutic strategy for GC patients with cisplatin resistance.

| S-EPMC10400920 | biostudies-literature

The novel ATR inhibitor VE-821 increases sensitivity of pancreatic cancer cells to radiation and chemotherapy.

Project description:DNA damaging agents such as radiotherapy and gemcitabine are frequently used for the treatment of pancreatic cancer. However, these treatments typically provide only modest benefit. Improving the low survival rate for pancreatic cancer patients therefore remains a major challenge in oncology. Inhibition of the key DNA damage response kinase ATR has been suggested as an attractive approach for sensitization of tumor cells to DNA damaging agents, but specific ATR inhibitors have remained elusive. Here we investigated the sensitization potential of the first highly selective and potent ATR inhibitor, VE-821, in vitro. VE-821 inhibited radiation- and gemcitabine-induced phosphorylation of Chk1, confirming inhibition of ATR signaling. Consistently, VE-821 significantly enhanced the sensitivity of PSN-1, MiaPaCa-2 and primary PancM pancreatic cancer cells to radiation and gemcitabine under both normoxic and hypoxic conditions. ATR inhibition by VE-821 led to inhibition of radiation-induced G 2/M arrest in cancer cells. Reduced cancer cell radiosurvival following treatment with VE-821 was also accompanied by increased DNA damage and inhibition of homologous recombination repair, as evidenced by persistence of γH2AX and 53BP1 foci and inhibition of Rad51 foci, respectively. These findings support ATR inhibition as a novel approach to improve the efficacy and therapeutic index of standard cancer treatments across a large proportion of pancreatic cancer patients.

| S-EPMC3461814 | biostudies-literature

Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition

Project description:Excessive genomic instability coupled with abnormalities in DNA repair pathways induce high levels of “replication stress” when cancer cells propagate. Rather than hampering cancer cell proliferation, novel treatment strategies are turning their attention toward targeting cell cycle checkpoint kinases (such as ATR, CHK1, WEE1 and others) along the DNA damage response and replicative stress response pathways, thereby allowing unrepaired DNA damage to be carried forward towards mitotic catastrophe and apoptosis. The selective inhibitor of ATR kinase elimusertib (BAY 1895344), has demonstrated preclinical and clinical monotherapy activity; however, reliable predictive biomarkers of treatment benefit are still lacking. In this study, using gene expression profiling of 24 cell lines from different cancer types and in a panel of ovarian cancer cell lines, we found that nuclear-specific enrichment of checkpoint kinase 1 (CHK1) correlated with increased sensitivity to elimusertib. Using an advanced multispectral imaging system in subsequent cell line-derived xenograft specimens, we showed a trend between nuclear phosphorylated-CHK1 (pCHK1) staining and increased sensitivity to the ATR inhibitor elimusertib, indicating the potential value of pCHK1 expression as a predictive biomarker of ATR inhibitor sensitivity.

2022-11-09 | GSE217579 | GEO

IRF3 drives replication stress signaling and determines sensitivity to genotoxic agents in triple-negative breast cancer cells

Project description:Interferon-β signaling has been shown to play important roles in anti-virus as well as anti-tumor immunity. We here report that the key transcription factor in type I interferon signaling, IRF3, as well as downstream interferon-β are able to induce replication stress signaling in triple-negative breast cancer cells. IRF3 overexpression sensitizes triple-negative breast cancer cells to several genotoxic agents. Addition of IFN-β, a key downstream cytokine of IRF3,reduced replication fork speed and activated ATR-CHK1 signaling. Conversely, type I interferon-neutralizing antibodies or nucleosides supplementation rescued IRF3-induced replication stress.

2024-05-01 | GSE263947 | GEO

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