ATR-Chk1 Pathway Research Articles

Base-excision repair pathway regulates transcription-replication conflicts in pancreatic ductal adenocarcinoma

Oncogenic mutations (such as in KRAS) can dysregulate transcription and replication, leading to transcription-replication conflicts (TRCs). Here, we demonstrate that TRCs are enriched in human pancreatic ductal adenocarcinoma (PDAC) compared to other common solid tumors or normal cells. Several orthogonal approaches demonstrated that TRCs are oncogene dependent. A small interfering RNA (siRNA) screen identified several factors in the base-excision repair (BER) pathway as main regulators of TRCs in PDAC cells. Inhibitors of BER pathway (methoxyamine and CRT) enhanced TRCs. Mechanistically, BER pathway inhibition severely altered RNA polymerase II (RNAPII) and R-loop dynamics at nascent DNA, causing RNAPII trapping and contributing to enhanced TRCs. The ensuing DNA damage activated the ATR-Chk1 pathway. Co-treatment with ATR inhibitor (VX970) and BER inhibitor (methoxyamine) at clinically relevant doses synergistically enhanced DNA damage and reduced cell proliferation in PDAC cells. The study provides mechanistic insights into the regulation of TRCs in PDAC by the BER pathway, which has biologic and therapeutic implications.

Synthetic lethality between ATR and POLA1 reveals a potential new target for individualized cancer therapy

The ATR-CHK1 pathway plays a fundamental role in the DNA damage response and is therefore an attractive target in cancer therapy. The antitumorous effect of ATR inhibitors is at least partly caused by synthetic lethality between ATR and various DNA repair genes. In previous studies, we have identified members of the B-family DNA polymerases as potential lethal partner for ATR, i.e. POLD1 and PRIM1. In this study, we validated and characterized the synthetic lethality between ATR and POLA1.First, we applied a model of ATR-deficient DLD-1 human colorectal cancer cells to confirm synthetic lethality by using chemical POLA1 inhibition. Analyzing cell cycle and apoptotic markers via FACS and Western blotting, we were able to show that apoptosis and S phase arrest contributed to the increased sensitivity of ATR-deficient cancer cells towards POLA1 inhibitors. Importantly, siRNA-mediated POLA1 depletion in ATR-deficient cells caused similar effects in regard to impaired cell viability and cumulation of apoptotic markers, thus excluding toxic effects of chemical POLA1 inhibition. Conversely, we demonstrated that siRNA-mediated POLA1 depletion sensitized several cancer cell lines towards chemical inhibition of ATR and its main effector kinase CHK1.In conclusion, the synthetic lethality between ATR/CHK1 and POLA1 might represent a novel and promising approach for individualized cancer therapy: First, alterations of POLA1 could serve as a screening parameter for increased sensitivity towards ATR and CHK1 inhibitors. Second, alterations in the ATR-CHK1 pathway might predict in increased sensitivity towards POLA1 inhibitors.

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.

Exacerbated Inflammatory Gene Expression After Impaired G2/M-Checkpoint Arrest in Fibroblasts Derived From a Patient Exhibiting Severe Adverse Effects

Purpose: Recent radiotherapy (RT), such as intensity-modulated radiation therapy and particle RT, has improved the concentration of the radiation field targeting tumors. However, severe adverse effects still occur, possibly due to genetic factors in patients. We aimed to investigate the mechanism of exacerbated inflammation during RT. Methods and MaterialsDermal fibroblasts derived from a patient with severe inflammatory adverse effects during RT were compared with two normal human dermal fibroblasts. Micronuclei formation, G2/M-checkpoint arrest, DNA damage signaling and repair, and inflammatory gene expression were comprehensively examined. Results: We found greater micronuclei formation in radiation-sensitive fibroblasts (RS-Fs) after ionizing radiation (IR). Importantly, RS-Fs exhibited premature G2/M-checkpoint release after IR, which triggers micronuclei formation because RS-Fs undergo mitosis with unrepaired DNA double-strand breaks (DSBs). Additionally, we found that DSB end-resection and activation of the ATR-Chk1 pathway were impaired in RS-Fs after IR. Consistent with the increase in the formation of micronuclei, which can deliver cytosolic nucleic acids resulting in an innate immune response, the expression of genes associated with inflammatory responses was highly upregulated in RS-Fs after IR. Conclusions: Although this is a single case of RT-dependent adverse effect, our findings suggest that impaired G2/M-checkpoint arrest due to the lack of DSB end-resection and activation of the ATR-Chk1 pathway causes exacerbated inflammation during RT; therefore, genes involved in G2/M-checkpoint arrest may be a predictive marker for unexpected inflammatory responses in RT.

ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia

BackgroundIn recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold‑inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC).MethodsCCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot.ResultsOur results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)‑like population. Moreover, hyperthermia substantially improved the sensitivity of radiation‑resistant NPC cells and CSC‑like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti‑tumor‑killing activity of hyperthermia against NPC cells and CSC‑like cells, whereas ectopic expression of Cirbp compromised tumor‑killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC‑like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance.ConclusionTaken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

A novel HDAC6 inhibitor interferes microtubule dynamics and spindle assembly checkpoint and sensitizes cisplatin-induced apoptosis in castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 μM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 μM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.

Structural basis for intra- and intermolecular interactions on RAD9 subunit of 9-1-1 checkpoint clamp implies functional 9-1-1 regulation by RHINO

Eukaryotic DNA clamp is a trimeric protein featuring a toroidal ring structure that binds DNA on the inside of the ring and multiple proteins involved in DNA transactions on the outside. Eukaryotes have two types of DNA clamps: the replication clamp PCNA and the checkpoint clamp RAD9-RAD1-HUS1 (9-1-1). 9-1-1 activates the ATR-CHK1 pathway in DNA damage checkpoint, regulating cell cycle progression. Structure of 9-1-1 consists of two moieties: a hetero-trimeric ring formed by PCNA-like domains of three subunits and an intrinsically disordered C-terminal region of the RAD9 subunit, called RAD9 C-tail. The RAD9 C-tail interacts with the 9-1-1 ring and disrupts the interaction between 9-1-1 and DNA, suggesting a negative regulatory role for this intramolecular interaction. In contrast, RHINO, a 9-1-1 binding protein, interacts with both RAD1 and RAD9 subunits, positively regulating checkpoint activation by 9-1-1. This study presents a biochemical and structural analysis of intra- and inter-molecular interactions on the 9-1-1 ring. Biochemical analysis indicates that RAD9 C-tail binds to the hydrophobic pocket on the PCNA-like domain of RAD9, implying that the pocket is involved in multiple protein-protein interactions. The crystal structure of the 9-1-1 ring in complex with a RHINO peptide reveals that RHINO binds to the hydrophobic pocket of RAD9, shedding light on the RAD9-binding motif. Additionally, the study proposes a structural model of the 9-1-1-RHINO quaternary complex. Together, these findings provide functional insights into the intra- and inter-molecular interactions on the front side of RAD9, elucidating the roles of RAD9 C-tail and RHINO in checkpoint activation.

Abstract B056: Modulation of the DNA replication stress response and chemo-response by the mono-ADP-ribosyltransferase PARP14

Abstract Genomic instability is a major hallmark of cancer and one of the most common causes is DNA replication stress. Mutations in genes involved in the DNA replication stress response are closely linked to cancer predisposition and progression but this has been exploited clinically. In the advent of personalized medicine, it has become clear that a better understanding of underlying mechanisms is important to advance cancer therapy, especially to address chemoresistance. Here, we identified novel roles for PARP14, a lesser studied member of the PARP family, in regulation of the replication stress response, and subsequently genomic stability and chemo-response. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we first catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Furthermore, we showed that PARP14 plays a central role in stabilization of stalled replication forks which arise due to high replication stress level in BRCA1/2 deficient cells. Mechanistically, we showed that PARP14 mediates stalled replication fork degradation via its interaction with the nuclease MRE11. Moreover, we also identified KU as an essential component of a multi-step mechanism by which the KU-PARP14 axis differentially promotes degradation by the nucleases MRE11 and EXO1. This mechanism restores genomic stability in BRCA-deficient cells and subsequently promotes chemoresistance. Overall, our work provides insights into novel mechanisms by which PARP14 affects the replication stress response and demonstrates that PARP14 can be used as a biomarker for chemo-response. Citation Format: Ashna Dhoonmoon, Claudia M Nicolae, George-Lucian Moldovan. Modulation of the DNA replication stress response and chemo-response by the mono-ADP-ribosyltransferase PARP14 [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 B056.

Investigation of ATR Inhibitor VX970 as a Radiosensitizer in Colorectal Cancer Cells

Colorectal cancer (CRC) is the second leading cause of combined cancer-related mortality in males and females in the U.S. Traditional treatment of locally advanced rectal cancer consists of neoadjuvant chemoradiation followed by surgery and adjuvant chemotherapy. Emerging data suggests that higher response rates can be achieved with total neoadjuvant therapy (TNT) where delivery of all chemotherapy and radiation therapy (RT) occurs prior to surgery. In addition, for patients with a complete response to TNT, non-operative management (NOM) can be considered. However, despite the use of TNT, pathologic complete response rates remain below 40% and NOM is only achieved in approximately 50% of rectal cancer patients. A strong need remains for more active anti-cancer therapies in rectal cancer to both reduce pelvic recurrence and facilitate NOM. Here, we tested the hypothesis that inhibition of the ataxia telangiectasia and Rad3-related protein kinase (ATR), a critical regulator of cellular DNA damage response, could increase the sensitivity of CRC to RT. VX970, a highly potent and selective ATR inhibitor, was investigated as a radiosensitizer in SW48 and LoVo CRC cell models. In vitro, IC50 of VX970 was assessed by alamarBlue cytotoxicity assay, while radiosensitivity was revealed by radiation clonogenic assays (0, 2, 4, 6, 8 Gy). ATR activity was determined by p-Chk1 using immunoblotting, and cell cycle distribution was analyzed by propidium iodide flow cytometry. CRC xenografts were generated using both LoVo and SW48 cells injected in the left flanks of athymic nude mice to explore the radiosensitizing effects of VX970 in vivo. Tumors were allowed to grow to 100-150 mm3, and the mice were randomized into multiple groups [vehicle alone, RT alone (10 Gy/5 fractions), VX970 alone, and VX970+RT]. Mouse weights and tumor size were measured three times weekly. Comparison of treatment groups was performed using the log-rank test with P<0.05 considered significant. The IC50 concentrations of VX970 on SW48 and LoVo cells were about 500 and 100 nM, respectively. VX970 at doses of 3 nM did not alter the viability of CRC cells, but significantly sensitized CRC cells to radiotherapy (P<0.05), with DER of 1.43 and 1.59, respectively, in SW48 and LoVo cells. VX970 efficiently attenuated p-Chk1 expression and significantly abrogated radiation induced G2/M cell cycle arrest (P<0.05). In addition, VX970 in combination with radiotherapy significantly prolonged tumor growth delay of CRC xenografts compared to radiation alone (P<0.05), with minimal toxicity observed. Inhibition of the ATR-Chk1 pathway by targeting ATR kinase with VX970 sensitizes CRC to radiotherapy in vitro and in vivo. Our findings support that ATR inhibition by VX970 is a promising new approach to improve the therapeutic ratio of radiotherapy for patients with CRC and warrants further clinical testing.

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

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.

Involvement of ATR-CHK1 pathway in fish megalocytivirus infection induced DNA-damage response in vitro

Increasing evidence has demonstrated that megalocytiviruses cause severe disease with high mortality in both freshwater and marine species. However, the molecular events employed in megalocytivirus infection in vitro still remained largely uncertain. Here, using an in vitro infection model of sea perch iridovirus (SPIV), we investigated the transcriptomic profiles of sea perch fry (SPF) cells upon SPIV infection, and elucidated the roles of crucial molecular events during infection using the specific inhibitors. Our data showed that almost all the differentially expressed genes (DEGs) associated with DNA-damage response (DDR), including DNA replication, mismatch repair, homologous recombination, nucleotide excision repair and Fanconi anemia (FA) pathway exhibited the up-regulation expression patterns during SPIV infection. As expected, DNA damage was detected in infected SPF cells, as evidenced by the DNA migration and the increased phosphorylation levels of H2AX along with the infection time. Furthermore, the key components of DDR, ataxia telangiectasia mutated and Rad3 related (ATR) and checkpoint kinase 1 (CHK1) kinases were elucidated to be essential for SPIV replication. Meanwhile, we also found that SPIV infection induced cell cycle arrest at G2/M phase, and 45 genes related to cell cycle pathway were up-regulated during infection. Inhibition of ATR activity induced cell cycle arrest of the infected cells at S phase, perhaps leading to the inhibitory effects on SPIV replication. Together, our data demonstrated for the first time that fish megalocytivirus induced DDR in vitro, and the ATR-CHK1 pathway exerted crucial roles in viral replication via regulating the cell cycle progression. These findings also provided new insights into the understanding of the interactions between fish megalocytivirus and host cells in vitro.

The MRN complex maintains the biliary-derived hepatocytes in liver regeneration through ATR-Chk1 pathway

When the proliferation of residual hepatocytes is prohibited, biliary epithelial cells (BECs) transdifferentiate into nascent hepatocytes to accomplish liver regeneration. Despite significant interest in transdifferentiation, little is known about the maintenance of nascent hepatocytes in post-injured environments. Here, we perform an N-ethyl-N-nitrosourea (ENU) forward genetic screen and identify a mutant containing a nonsense mutation in the gene nibrin (nbn), which encodes a component of the Mre11-Rad50-Nbn (MRN) complex that activates DNA damage response (DDR). The regenerated hepatocytes cannot be maintained and exhibit apoptosis in the mutant. Mechanistically, the nbn mutation results in the abrogation of ATR-Chk1 signaling and accumulations of DNA damage in nascent hepatocytes, which eventually induces p53-mediated apoptosis. Furthermore, loss of rad50 or mre11a shows similar phenotypes. This study reveals that the activation of DDR by the MRN complex is essential for the survival of BEC-derived hepatocytes, addressing how to maintain nascent hepatocytes in the post-injured environments.

RNF126-Mediated MRE11 Ubiquitination Activates the DNA Damage Response and Confers Resistance of Triple-Negative Breast Cancer to Radiotherapy.

Triple-negative breast cancer (TNBC) has higher molecular heterogeneity and metastatic potential and the poorest prognosis. Because of limited therapeutics against TNBC, irradiation (IR) therapy is still a common treatment option for patients with lymph nodes or brain metastasis. Thus, it is urgent to develop strategies to enhance the sensitivity of TNBC tumors to low-dose IR. Here, the authors report that E3 ubiquitin ligase Ring finger protein 126 (RNF126) is important for IR-induced ATR-CHK1 pathway activation to enhance DNA damage repair (DDR). Mechanistically, RNF126 physically associates with the MRE11-RAD50-NBS1 (MRN) complex and ubiquitinates MRE11 at K339 and K480 to increase its DNA exonuclease activity, subsequent RPA binding, and ATR phosphorylation, promoting sustained DDR in a homologous recombination repair-prone manner. Accordingly, depletion of RNF126 leads to increased genomic instability and radiation sensitivity in both TNBC cells and mice. Furthermore, it is found that RNF126 expression is induced by IR activating the HER2-AKT-NF-κB pathway and targeting RNF126 expression with dihydroartemisinin significantly improves the sensitivity of TNBC tumors in the brain to IR treatment in vivo. Together, these results reveal that RNF126-mediated MRE11 ubiquitination is a critical regulator of the DDR, which provides a promising target for improving the sensitivity of TNBC to radiotherapy.

ML216-Induced BLM Helicase Inhibition Sensitizes PCa Cells to the DNA-Crosslinking Agent Cisplatin.

Using standard DNA-damaging medicines with DNA repair inhibitors is a promising anticancer tool to achieve better therapeutic responses and reduce therapy-related side effects. Cell viability assay, neutral comet assay, western blotting (WB), and cell cycle and apoptosis analysis were used to determine the synergistic effect and mechanism of ML216, a Bloom syndrome protein (BLM) helicase inhibitor, and cisplatin (CDDP), a DNA-crosslinking agent, in PCa cells. Based on the online database research, our findings revealed that BLM was substantially expressed in PCa, which is associated with a bad prognosis for PCa patients. The combination of ML216 and CDDP improved the antiproliferative properties of three PCa cell lines. As indicated by the increased production of γH2AX and caspase-3 cleavage, ML216 significantly reduced the DNA damage-induced high expression of BLM, making PC3 more susceptible to apoptosis and DNA damage caused by CDDP. Furthermore, the combination of ML216 and CDDP increased p-Chk1 and p-Chk2 expression. The DNA damage may have triggered the ATR-Chk1 and ATM-Chk2 pathways simultaneously. Our results demonstrated that ML216 and CDDP combination therapy exhibited synergistic effects, and combination chemotherapy could be a novel anticancer tactic.

Inhibition of Chk2 promotes neuroprotection, axon regeneration, and functional recovery after CNS injury.

DNA double-strand breaks occur in many acute and long-term neurological conditions, including neurodegeneration, neurotrauma, and stroke. Nonrepaired breaks chronically activate the DNA damage response in neurons, leading to neural dysfunction and apoptosis. Here, we show that targeting of the central ATM-Chk2 pathway regulating the response to double-strand breaks slows neural decline in Drosophila models of chronic neurodegeneration. Inhibitors of ATM-Chk2, but not the parallel ATR-Chk1 pathway, also promote marked, functional recovery after acute central nervous system injury in rats, suggesting that inhibiting nonhomologous end-joining rather than homologous recombination is crucial for neuroprotection. We demonstrate that the Chk2 inhibitor, prexasertib, which has been evaluated in phase 2 clinical trials for cancer, has potent neuroprotective effects and represents a new treatment option to promote functional recovery after spinal cord or optic nerve injury.

A clinically relevant heterozygous ATR mutation sensitizes colorectal cancer cells to replication stress

Colorectal cancer (CRC) ranks third among the most frequent malignancies and represents the second most common cause of cancer-related deaths worldwide. By interfering with the DNA replication process of cancer cells, several chemotherapeutic molecules used in CRC therapy induce replication stress (RS). At the cellular level, this stress is managed by the ATR-CHK1 pathway, which activates the replication checkpoint. In recent years, the therapeutic value of targeting this pathway has been demonstrated. Moreover, MSI + (microsatellite instability) tumors frequently harbor a nonsense, heterozygous mutation in the ATR gene. Using isogenic HCT116 clones, we showed that this mutation of ATR sensitizes the cells to several drugs, including SN-38 (topoisomerase I inhibitor) and VE-822 (ATR inhibitor) and exacerbates their synergistic effects. We showed that this mutation bottlenecks the replication checkpoint leading to extensive DNA damage. The combination of VE-822 and SN-38 induces an exhaustion of RPA and a subsequent replication catastrophe. Surviving cells complete replication and accumulate in G2 in a DNA-PK-dependent manner, protecting them from cell death. Together, our results suggest that RPA and DNA-PK represent promising therapeutic targets to optimize the inhibition of the ATR-CHK1 pathway in oncology. Ultimately, ATR frameshift mutations found in patients may also represent important prognostic factors.

ACTL6A deficiency induces apoptosis through impairing DNA replication and inhibiting the ATR-Chk1 signaling in glioblastoma cells

Actin-like 6A (ACTL6A) is a core subunit of the SWI/SNF chromatin remodeling complex and is highly expressed in several types of human cancers including glioblastoma. Recent studies verified that ACTL6A regulates the proliferation, differentiation, and migration of cancer cells. In this study, we identified ACTL6A as an important regulator of DNA replication. ACTL6A knockdown could impair the DNA replication initiation in glioblastoma cells. The regulation of DNA replication by ACTL6A was mediated through regulating the expression of the CDC45-MCM-GINS (CMG) complex genes. Further investigation revealed that ACTL6A transcriptionally regulates MCM5 expression. Furthermore, ACTL6A knockdown induced DNA damage and diminished the activity of the ATR-Chk1 pathway, which ultimately led glioblastoma cells to apoptosis and death. Taken together, our findings highlight the critical role of ACTL6A in DNA replication and ATR-Chk1 pathway, and reveal a potential target for therapeutic intervention in glioblastoma.

A Tale of Two Checkpoints: ATR Inhibition and PD-(L)1 Blockade

Innate immunity and the DNA damage response (DDR) pathway are inextricably linked. Within the DDR, ataxia telangiectasia and Rad3-related (ATR) is a key kinase responsible for sensing replication stress and facilitating DNA repair through checkpoint activation, cell cycle arrest, and promotion of fork recovery. Recent studies have shed light on the immunomodulatory role of the ATR-CHK1 pathway in the tumor microenvironment and the specific effects of ATR inhibition in stimulating an innate immune response. With several potent and selective ATR inhibitors in developmental pipelines, the combination of dual ATR and PD-(L)1 blockade has attracted increasing interest in cancer therapy. In this review, we summarize the clinical and preclinical data supporting the combined inhibition of ATR and PD-(L)1, discuss the potential challenges surrounding this approach, and highlight biomarkers relevant for selected patients who are most likely to benefit from the blockade of these two checkpoints.

ZGRF1 promotes end resection of DNA homologous recombination via forming complex with BRCA1/EXO1

To maintain genomic stability, the mammalian cells has evolved a coordinated response to DNA damage, including activation of DNA repair and cell cycle checkpoint processes. Exonuclease 1 (EXO1)-dependent excision of DNA ends is important for the initiation of homologous recombination (HR) repair of DNA breaks, which is thought to play a key role in activating the ATR-CHK1 pathway to induce G2/M cell cycle arrest. But the mechanism is still not fully understood. Here, we report that ZGRF1 forms complexes with EXO1 as well as other repair proteins and promotes DNA repair through HR. ZGRF1 is recruited to DNA damage sites in a MDC1-RNF8-BRCA1 dependent manner. Furthermore, ZGRF1 is important for the recruitment of RPA2 to DNA damage sites and the following ATR-CHK1 mediated G2/M checkpoint in response to irradiation. ZGRF1 null cells show increased sensitivity to many DNA-damaging agents, especially PARPi and irradiation. Collectively,our findings identify ZGRF1 as a novel regulator of DNA end resection and G2/M checkpoint. ZGRF1 is a potential target of radiation and PARPi cancer therapy.

Exploring the ATR-CHK1 pathway in the response of doxorubicin-induced DNA damages in acute lymphoblastic leukemia cells

Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B−/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B−/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses.Graphical abstract• Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells.• ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway.• The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells.• The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.