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In autosomal dominant polycystic kidney disease (ADPKD) with germline mutations in a PKD1 or PKD2 gene, innumerable cysts develop from tubules, and renal function deteriorates. Second-hit somatic mutations and renal tubular epithelial (RTE) cell death are crucial features of cyst initiation and disease progression. Here, we use established RTE lines and primary ADPKD cells with disease-associated PKD1 mutations to investigate genomic instability and DNA damage responses. We found that ADPKD cells suffer severe chromosome breakage, aneuploidy, heightened susceptibility to DNA damage, and delayed checkpoint activation. Immunohistochemical analyses of human kidneys corroborated observations in cultured cells. DNA damage sensors (ATM/ATR) were activated but did not localize at nuclear sites of damaged DNA and did not properly activate downstream transducers (CHK1/CHK2). ADPKD cells also had the ability to transform, as they achieved high saturation density and formed colonies in soft agar. Our studies indicate that defective DNA damage repair pathways and the somatic mutagenesis they cause contribute fundamentally to the pathogenesis of ADPKD. Acquired mutations may alternatively confer proliferative advantages to the clonally expanded cell populations or lead to apoptosis. Further understanding of the molecular details of aberrant DNA damage responses in ADPKD is ongoing and holds promise for targeted therapies.

Simple Summary: Around 50% of all human cancers are treated with radiotherapy. The effectiveness of radiotherapy is driven through causing DNA damage within the cancer cells; however, the cells respond by activating repair mechanisms that can lead to resistance to treatment. A promising strategy is to target one of these defence mechanisms, called the cell cycle checkpoint, using specific drugs/inhibitors that can be used in combination with radiotherapy. Here, we review evidence of investigations into inhibitors of two important proteins (Chk1 and Wee1) and how these can be used to increase the effectiveness of radiotherapy in cancer treatment. Radiotherapy is one of the main cancer treatments being used for ~50% of all cancer patients. Conventional radiotherapy typically utilises X-rays (photons); however, there is increasing use of particle beam therapy (PBT), such as protons and carbon ions. This is because PBT elicits significant benefits through more precise dose delivery to the cancer than X-rays, but also due to the increases in linear energy transfer (LET) that lead to more enhanced biological effectiveness. Despite the radiotherapy type, the introduction of DNA damage ultimately drives the therapeutic response through stimulating cancer cell death. To combat this, cells harbour cell cycle checkpoints that enables time for efficient DNA damage repair. Interestingly, cancer cells frequently have mutations in key genes such as TP53 and ATM that drive the G1/S checkpoint, whereas the G2/M checkpoint driven through ATR, Chk1 and Wee1 remains intact. Therefore, targeting the G2/M checkpoint through specific inhibitors is considered an important strategy for enhancing the efficacy of radiotherapy. In this review, we focus on inhibitors of Chk1 and Wee1 kinases and present the current biological evidence supporting their utility as radiosensitisers with different radiotherapy modalities, as well as clinical trials that have and are investigating their potential for cancer patient benefit. [ABSTRACT FROM AUTHOR]

Multidrug resistance is a substantial obstacle in treating non-small cell lung cancer (NSCLC) with therapies like cisplatin (DDP)-based adjuvant chemotherapy and EGFR-tyrosine kinase inhibitors (TKIs). Aaptamine-7 (AP-7), a benzonaphthyridine alkaloid extracted from Aaptos aaptos sponge, has been shown to exhibit a broad spectrum of anti-tumor activity. However, the anticancer activity of AP-7 in combination with DDP and its molecular mechanisms in multidrug-resistant NSCLC are not yet clear. Our research indicates that AP-7 bolsters the growth inhibition activity of DDP on multidrug-resistant NSCLC cells. AP-7 notably disrupts DDP-induced cell cycle arrest and amplifies DDP-induced DNA damage effects in these cells. Furthermore, the combination of AP-7 and DDP downregulates Chk1 activation, interrupts the DNA damage repairdependent Chk1/CDK1 pathway, and helps to overcome drug resistance and boost apoptosis in multidrug-resistant NSCLC cells and a gefitinib-resistant xenograft mice model. In summary, AP-7 appears to enhance DDP-induced DNA damage by impeding the Chk1 signaling pathway in multidrug-resistant NSCLC, thereby augmenting growth inhibition, both in vitro and in vivo. These results indicate the potential use of AP-7 as a DDP sensitizer in the treatment of multidrug-resistant NSCLC. [ABSTRACT FROM AUTHOR]

The ATR-Chk1 pathway is essential in cellular responses to DNA damage and replication stress, whereas the role of long noncoding RNAs (lncRNAs) in regulating this pathway remains largely unknown. In this study, we identify an ATR and Chk1 interacting lncRNA (ACIL, also known as LRRC75A-AS1 or SNHG29), which promotes the phosphorylation of Chk1 by ATR upon DNA damages. High ACIL levels are associated with chemoresistance to DNA damaging agents and poor outcome of breast cancer patients. ACIL knockdown sensitizes breast cancer cells to DNA damaging drugs in vitro and in vivo. ACIL protects cancer cells against DNA damages by inducing cell cycle arrest, stabilizing replication forks and inhibiting unscheduled origin firing, thereby guarding against replication catastrophe and contributing to DNA damage repair. These findings demonstrate a lncRNA-dependent mechanism of activating the ATR-Chk1 pathway and highlight the potential of utilizing ACIL as a predictive biomarker for chemotherapy sensitivity, as well as targeting ACIL to reverse chemoresistance in breast cancer.

Checkpoint kinase 1 (Chk1) is a key mediator of the DNA damage response that regulates cell cycle progression, DNA damage repair, and DNA replication. Small-molecule Chk1 inhibitors sensitize cancer cells to genotoxic agents and have shown preclinical activity as single agents in cancers characterized by high levels of replication stress. However, the underlying genetic determinants of Chk1-inhibitor sensitivity remain unclear. Although treatment options for advanced colorectal cancer are limited, radiotherapy is effective. Here, we report that exposure to a novel amidine derivative, K1586, leads to an initial reduction in the proliferative potential of colorectal cancer cells. Cell cycle analysis revealed that the length of the G2/M phase increased with K1586 exposure as a result of Chk1 instability. Exposure to K1586 enhanced the degradation of Chk1 in a time- and dose-dependent manner, increasing replication stress and sensitizing colorectal cancer cells to radiation. Taken together, the results suggest that a novel amidine derivative may have potential as a radiotherapy-sensitization agent that targets Chk1. [ABSTRACT FROM AUTHOR]

Background: VE-822 is a novel inhibitor of ATR, a key kinase involved in the DNA damage response pathway. The role of ATR inhibition in reversing drug resistance in various cancer types has been investigated. Therefore, this study investigated the effects of ATR inhibition by VE-822 on reversing 5-fluorouracil (5-FU) resistance in colorectal cancer cell line (Caco-2). Methods: Caco-2 and 5-FU resistance Caco-2 (Caco-2/5-FU) cells were treated with 5-FU and VE-822, alone and in combination. Cell proliferation and viability were assessed by MTT assay and Trypan Blue staining. P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) activities were measured by Rhodamine123 accumulation and uptake assay. The mRNA levels of P-gp, MRP-1, ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 1 (CHK1) were measured by qRT-PCR. Western blot was used to measure the protein levels of P-gp, MRP-1, γ-H2AX, ATR and CHK1 in cells. 8-Oxo-2'-deoxyguanosine (8-oxo-dG) levels were determined via ELISA. Apoptosis was evaluated by ELISA death assay, DAPI staining and lactate dehydrogenase (LDH) assay. Results: The Caco-2/5-FU cells showed lower levels of 5-FU mediated proliferation inhibition in comparison to Caco-2 cells. VE-822 decreased the IC50 value of 5-FU on resistant cells. In addition, the expression levels and activity of P-gp and MRP-1 were significantly decreased in resistant cells treated with VE-822 (P < 0.05). The combination of 5-FU and VE-822 increased apoptosis in Caco-2/5-FU cells by downregulating CHK1 and ATR and upregulating γ-H2AX and 8-oxo-dG. Conclusion: The simultaneous treatment of resistant colorectal cancer cells with 5-FU and ATR inhibitor, VE-822, was demonstrated to be effective in reversing drug resistance and potentiating 5-FU mediated anticancer effects via targeting DNA damage. [ABSTRACT FROM AUTHOR]

Replication stress (RS) is a characteristic state of cancer cells as they tend to exchange precision of replication for fast proliferation and increased genomic instability. To overcome the consequences of improper replication control, malignant cells frequently inactivate parts of their DNA damage response (DDR) pathways (the ATM-CHK2-p53 pathway), while relying on other pathways which help to maintain replication fork stability (ATR-CHK1). This creates a dependency on the remaining DDR pathways, vulnerability to further destabilization of replication and synthetic lethality of DDR inhibitors with common oncogenic alterations such as mutations of TP53, RB1, ATM, amplifications of MYC, CCNE1 and others. The response to RS is normally limited by coordination of cell cycle, transcription and replication. Inhibition of WEE1 and PKMYT1 kinases, which prevent unscheduled mitosis entry, leads to fragility of under-replicated sites. Recent evidence also shows that inhibition of Cyclin-dependent kinases (CDKs), such as CDK4/6, CDK2, CDK8/19 and CDK12/13 can contribute to RS through disruption of DNA repair and replication control. Here, we review the main causes of RS in cancers as well as main therapeutic targets—ATR, CHK1, PARP and their inhibitors. [ABSTRACT FROM AUTHOR]

Checkpoint kinase 1 (CHK1), a serine/threonine kinase, plays a crucial role in cell cycle arrest and is a promising therapeutic target for drug development against cancers. CHK1 coordinates cell cycle checkpoints in response to DNA damage, facilitating repair of single-strand breaks, and maintains the genome integrity in response to replication stress. In this study, we employed an integrated computational and experimental approach to drug discovery and repurposing, aiming to identify a potent CHK1 inhibitor among existing drugs. An e-pharmacophore model was developed based on the three-dimensional crystal structure of the CHK1 protein in complex with CCT245737. This model, characterized by seven key molecular features, guided the screening of a library of drugs through molecular docking. The top 10% of scored ligands were further examined, with procaterol emerging as the leading candidate. Procaterol demonstrated interaction patterns with the CHK1 active site similar to CHK1 inhibitor (CCT245737), as shown by molecular dynamics analysis. Subsequent in vitro assays, including cell proliferation, colony formation, and cell cycle analysis, were conducted on gastric adenocarcinoma cells treated with procaterol, both as a monotherapy and in combination with cisplatin. Procaterol, in synergy with cisplatin, significantly inhibited cell growth, suggesting a potentiated therapeutic effect. Thus, we propose the combined application of cisplatin and procaterol as a novel potential therapeutic strategy against human gastric cancer. The findings also highlight the relevance of CHK1 kinase as a drug target for enhancing the sensitivity of cytotoxic agents in cancer. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double)strand break induction, as evidenced by an increased percentage of gH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors’ combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression. [ABSTRACT FROM AUTHOR]

Inhibition of checkpoint kinase 1 (Chk1) has shown to overcome resistance to poly (ADP-ribose) polymerase (PARP) inhibitors and expand the clinical utility of PARP inhibitors in a broad range of human cancers. Pristimerin, a naturally occurring pentacyclic triterpenoid, has been the focus of intensive studies for its anticancer potential. However, it is not yet known whether low dose of pristimerin can be combined with PARP inhibitors by targeting Chk1 signaling pathway. In this study, we investigated the efficacy, safety and molecular mechanisms of the synergistic effect produced by the combination olaparib and pristimerin in TP53-deficient and BRCA-proficient cell models. As a result, an increased expression of Chk1 was correlated with TP53 mutation, and pristimerin preferentially sensitized p53-defective cells to olaparib. The combination of olaparib and pristimerin resulted in a more pronounced abrogation of DNA synthesis and induction of DNA double-strand breaks (DSBs). Moreover, pristimerin disrupted the constitutional levels of Chk1 and DSB repair activities. Mechanistically, pristimerin promoted K48-linked polyubiquitination and proteasomal degradation of Chk1 while not affecting its kinase domain and activity. Importantly, combinatorial therapy led to a higher rate of tumor growth inhibition without apparent hematological toxicities. In addition, pristimerin suppressed olaparib-induced upregulation of Chk1 and enhanced olaparib-induced DSB marker γΗ2ΑΧ in vivo. Taken together, inhibition of Chk1 by pristimerin has been observed to induce DNA repair deficiency, which may expand the application of olaparib in BRCA-proficient cancers harboring TP53 mutations. Thus, pristimerin can be combined for PARP inhibitor-based therapy.

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors’ combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression.

ATR kinase inhibitors promote cell killing by inducing replication stress and through potentiation of genotoxic agents in gynecologic cancer cells. To explore mechanisms of acquired resistance to ATRi in ovarian cancer, we characterized ATRi-resistant ovarian cancer cells generated by metronomic dosing with the clinical ATR inhibitor AZD6738. ATRi-resistant ovarian cancer cells (OVCAR3 and OV90) were generated by dosing with AZD6738 and assessed for sensitivity to Chk1i (LY2603618), PARPi (Olaparib) and combination with cisplatin or a CDK4/6 inhibitor (Palbociclib). Models were characterized by diverse methods including silencing CDC25A in OV90 cells and assessing impact on ATRi response. Serum proteomic analysis of ATRi-resistant OV90 xenografts was performed to identify circulating biomarker candidates of ATRi-resistance. AZD6738-resistant cell lines are refractory to LY2603618, but not to Olaparib or combinations with cisplatin. Cell cycle analyses showed ATRi-resistant cells exhibit G1/S arrest following AZD6738 treatment. Accordingly, combination with Palbociclib confers resistance to AZD6738. AZD6738-resistant cells exhibit altered abundances of G1/S phase regulatory proteins, including loss of CDC25A in AZD6738-resistant OV90 cells. Silencing of CDC25A in OV90 cells confers resistance to AZD6738. Serum proteomics from AZD6738-resistant OV90 xenografts identified Vitamin D-Binding Protein (GC), Apolipoprotein E (APOE) and A1 (APOA1) as significantly elevated in AZD6738-resistant backgrounds. We show that metronomic dosing of ovarian cancer cells with AZD6738 results in resistance to ATR/ Chk1 inhibitors, that loss of CDC25A expression represents a mechanism of resistance to ATRi treatment in ovarian cancer cells and identify several circulating biomarker candidates of CDC25A low, AZD6738-resistant ovarian cancer cells. [Display omitted] • Metronomic dosing of ovarian cancer cells with AZD6738 creates resistance to AZD6738 and LY2603618, but not to Olaparib. • AZD6738 resistant ovarian cancer cells remain sensitive to combination treatment with AZD6738 and CDDP. • Combination treatment of ovarian cancer cells with Palbociclib and AZD6738 promotes resistance to ATRi treatment. • Loss of CDC25A expression represents a mechanism of acquired resistance to AZD6738 treatment in ovarian cancer cells. • Xenograft mice with CDC25A low, ATRi-resistant ovarian tumors exhibit elevated levels of circulating GC, APOE and APOA1. [ABSTRACT FROM AUTHOR]

Locally advanced head and neck squamous cell carcinomas (HNSCC) are often refractory to platinum-based radiochemotherapy and new immuno-oncological strategies. To stimulate immunogenic antitumor responses in HNSCC patients, we investigated the cGAS/STING/IFN-1 signaling pathway after genotoxic treatments and concomitant abrogation of the DNA damage response (DDR). For this purpose, FaDu and UM-SCC1 cells were exposed to X-rays or cisplatin and treated with an ATR or Chk1 inhibitor, or by Fanconi anemia gene A knockout (FANCA ko). We assessed clonogenic survival, cell cycle regulation, micronuclei, free cytosolic double-stranded DNA, and the protein expression and activity of the cGAS/STING/IFN-1 pathway and related players. Cell survival, regulation of G2/M arrest, and formation of rupture-prone cGAS-positive micronuclei after genotoxic treatments were most affected by ATR inhibition and FANCA ko. In UM-SCC-1 cells only, 8 Gy X-rays promoted IFN-1 expression unaltered by abrogation of the DDR or concomitant increased TREX1 expression. At a higher dose of 20 Gy, this effect was observed only for concurrent Chk1- or ATR-inhibition. FANCA ko or cisplatin treatment was ineffective in this regard. Our observations open new perspectives for the enhancement of cGAS/STING/IFN-1-mediated antitumor immune response in HNSCC by hypofractionated or stereotactic radiotherapy concepts in multimodal settings with immuno-oncological strategies. [ABSTRACT FROM AUTHOR]

The DNA damage response (DDR) acts as a barrier to malignant transformation and is often impaired during tumorigenesis. Exploiting the impaired DDR can be a promising therapeutic strategy; however, the mechanisms of inactivation and corresponding biomarkers are incompletely understood. Starting from an unbiased screening approach, we identified the SMC5‐SMC6 Complex Localization Factor 2 (SLF2) as a regulator of the DDR and biomarker for a B‐cell lymphoma (BCL) patient subgroup with an adverse prognosis. SLF2‐deficiency leads to loss of DDR factors including Claspin (CLSPN) and consequently impairs CHK1 activation. In line with this mechanism, genetic deletion of Slf2 drives lymphomagenesis in vivo. Tumor cells lacking SLF2 are characterized by a high level of DNA damage, which leads to alterations of the post‐translational SUMOylation pathway as a safeguard. The resulting co‐dependency confers synthetic lethality to a clinically applicable SUMOylation inhibitor (SUMOi), and inhibitors of the DDR pathway act highly synergistic with SUMOi. Together, our results identify SLF2 as a DDR regulator and reveal co‐targeting of the DDR and SUMOylation as a promising strategy for treating aggressive lymphoma. Synopsis: SLF2 was identified as a tumor suppressor of B‐cell lymphomagenesis and a crucial regulator of CHK1, and its deficiency was associated with defective DNA damage response and synthetic lethality to SUMOylation inhibition. SLF2 is a functionally relevant tumor suppressor in murine and human B‐cell lymphoma.Loss of SLF2 results in CHK1 impairment, transcriptional or post‐transcriptional repression of Claspin, and genomic instability.SLF2 deficiency drives alteration of the SUMO pathway and confers synthetic lethality to pharmacological SUMOylation inhibition.Impaired DNA damage responses, e.g., caused by SLF2 loss or pharmacological inhibition of CHK1, confer synthetic lethality to SUMO inhibition. [ABSTRACT FROM AUTHOR]

Checkpoint kinase 1 (Chk1) is a key mediator of the DNA damage response that regulates cell cycle progression, DNA damage repair, and DNA replication. Small-molecule Chk1 inhibitors sensitize cancer cells to genotoxic agents and have shown preclinical activity as single agents in cancers characterized by high levels of replication stress. However, the underlying genetic determinants of Chk1-inhibitor sensitivity remain unclear. Although treatment options for advanced colorectal cancer are limited, radiotherapy is effective. Here, we report that exposure to a novel amidine derivative, K1586, leads to an initial reduction in the proliferative potential of colorectal cancer cells. Cell cycle analysis revealed that the length of the G2/M phase increased with K1586 exposure as a result of Chk1 instability. Exposure to K1586 enhanced the degradation of Chk1 in a time- and dose-dependent manner, increasing replication stress and sensitizing colorectal cancer cells to radiation. Taken together, the results suggest that a novel amidine derivative may have potential as a radiotherapy-sensitization agent that targets Chk1.

In autosomal dominant polycystic kidney disease (ADPKD) with germline mutations in a PKD1 or PKD2 gene, innumerable cysts develop from tubules, and renal function deteriorates. Second-hit somatic mutations and renal tubular epithelial (RTE) cell death are crucial features of cyst initiation and disease progression. Here, we use established RTE lines and primary ADPKD cells with disease-associated PKD1 mutations to investigate genomic instability and DNA damage responses. We found that ADPKD cells suffer severe chromosome breakage, aneuploidy, heightened susceptibility to DNA damage, and delayed checkpoint activation. Immunohistochemical analyses of human kidneys corroborated observations in cultured cells. DNA damage sensors (ATM/ATR) were activated but did not localize at nuclear sites of damaged DNA and did not properly activate downstream transducers (CHK1/CHK2). ADPKD cells also had the ability to transform, as they achieved high saturation density and formed colonies in soft agar. Our studies indicate that defective DNA damage repair pathways and the somatic mutagenesis they cause contribute fundamentally to the pathogenesis of ADPKD. Acquired mutations may alternatively confer proliferative advantages to the clonally expanded cell populations or lead to apoptosis. Further understanding of the molecular details of aberrant DNA damage responses in ADPKD is ongoing and holds promise for targeted therapies.

Abstract The DNA damage response (DDR) acts as a barrier to malignant transformation and is often impaired during tumorigenesis. Exploiting the impaired DDR can be a promising therapeutic strategy; however, the mechanisms of inactivation and corresponding biomarkers are incompletely understood. Starting from an unbiased screening approach, we identified the SMC5‐SMC6 Complex Localization Factor 2 (SLF2) as a regulator of the DDR and biomarker for a B‐cell lymphoma (BCL) patient subgroup with an adverse prognosis. SLF2‐deficiency leads to loss of DDR factors including Claspin (CLSPN) and consequently impairs CHK1 activation. In line with this mechanism, genetic deletion of Slf2 drives lymphomagenesis in vivo. Tumor cells lacking SLF2 are characterized by a high level of DNA damage, which leads to alterations of the post‐translational SUMOylation pathway as a safeguard. The resulting co‐dependency confers synthetic lethality to a clinically applicable SUMOylation inhibitor (SUMOi), and inhibitors of the DDR pathway act highly synergistic with SUMOi. Together, our results identify SLF2 as a DDR regulator and reveal co‐targeting of the DDR and SUMOylation as a promising strategy for treating aggressive lymphoma.

BackgroundThe deSUMOylase sentrin-specific isopeptidase 2 (SENP2) plays a crucial role in atheroprotection. However, the phosphorylation of SENP2 at T368 under disturbed flow (D-flow) conditions hinders its nuclear function and promotes endothelial cell (EC) activation. SUMOylation has been implicated in D-flow-induced endothelial-to-mesenchymal transition (endoMT), but the precise role of SENP2 in counteracting this process remains unclear.MethodWe developed a phospho-specific SENP2 S344 antibody and generated knock-in (KI) mice with a phospho-site mutation of SENP2 S344A using CRISPR/Cas9 technology. We then investigated the effects of SENP2 S344 phosphorylation under two distinct flow patterns and during hypercholesteremia (HC)-mediated EC activation.ResultOur findings demonstrate that laminar flow (L-flow) induces phosphorylation of SENP2 at S344 through the activation of checkpoint kinase 1 (CHK1), leading to the inhibition of ERK5 and p53 SUMOylation and subsequent suppression of EC activation. We observed a significant increase in lipid-laden lesions in both the aortic arch (under D-flow) and descending aorta (under L-flow) of female hypercholesterolemic SENP2 S344A KI mice. In male hypercholesterolemic SENP2 S344A KI mice, larger lipid-laden lesions were only observed in the aortic arch area, suggesting a weaker HC-mediated atherogenesis in male mice compared to females. Ionizing radiation (IR) reduced CHK1 expression and SENP2 S344 phosphorylation, attenuating the pro-atherosclerotic effects observed in female SENP2 S344A KI mice after bone marrow transplantation (BMT), particularly in L-flow areas. The phospho-site mutation SENP2 S344A upregulates processes associated with EC activation, including inflammation, migration, and proliferation. Additionally, fibrotic changes and up-regulated expression of EC marker genes were observed. Apoptosis was augmented in ECs derived from the lungs of SENP2 S344A KI mice, primarily through the inhibition of ERK5-mediated expression of DNA damage-induced apoptosis suppressor (DDIAS).SummaryIn this study, we have revealed a novel mechanism underlying the suppressive effects of L-flow on EC inflammation, migration, proliferation, apoptosis, and fibrotic changes through promoting CHK1-induced SENP2 S344 phosphorylation. The phospho-site mutation SENP2 S344A responds to L-flow through a distinct mechanism, which involves the upregulation of both mesenchymal and EC marker genes.

BackgroundDogs present a significant opportunity for studies in comparative oncology. However, the study of cancer biology phenomena in canine cells is currently limited by restricted availability of validated antibody reagents and techniques. Here, we provide an initial characterization of the expression and activity of key components of the DNA Damage Response (DDR) in a panel of hematopoietic canine cancer cell lines, with the use of commercially available antibody reagents.Materials and methodsThe techniques used for this validation analysis were western blot, qPCR, and DNA combing assay.ResultsSubstantial variations in both the basal expression (ATR, Claspin, Chk1, and Rad51) and agonist-induced activation (p-Chk1) of DDR components were observed in canine cancer cell lines. The expression was stronger in the CLBL-1 (B-cell lymphoma) and CLB70 (B-cell chronic lymphocytic leukemia) cell lines than in the GL-1 (B-cell leukemia) cell line, but the biological significance of these differences requires further investigation. We also validated methodologies for quantifying DNA replication dynamics in hematopoietic canine cancer cell lines, and found that the GL-1 cell line presented a higher replication fork speed than the CLBL-1 cell line, but that both showed a tendency to replication fork asymmetry.ConclusionThese findings will inform future studies on cancer biology, which will facilitate progress in developing novel anticancer therapies for canine patients. They can also provide new knowledge in human oncology.

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. • 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. [ABSTRACT FROM AUTHOR]

We have recently reported that in G2-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the G2-checkpoint epistatically, with ATR at the output-node, interfacing with the cell cycle through Chk1. However, although inhibition of ATR nearly completely abrogated the checkpoint, inhibition of Chk1 using UCN-01 generated only partial responses. This suggested that additional kinases downstream of ATR were involved in the transmission of the signal to the cell cycle engine. Additionally, the broad spectrum of kinases inhibited by UCN-01 pointed to uncertainties in the interpretation that warranted further investigations. Here, we show that more specific Chk1 inhibitors exert an even weaker effect on G2-checkpoint, as compared to ATR inhibitors and UCN-01, and identify the MAPK p38α and its downstream target MK2 as checkpoint effectors operating as backup to Chk1. These observations further expand the spectrum of p38/MK2 signaling to G2-checkpoint activation, extend similar studies in cells exposed to other DNA damaging agents and consolidate a role of p38/MK2 as a backup kinase module, adding to similar backup functions exerted in p53 deficient cells. The results extend the spectrum of actionable strategies and targets in current efforts to enhance the radiosensitivity in tumor cells. [ABSTRACT FROM AUTHOR]

Abstract Background Osteosarcoma is a malignant bone tumor that usually affects adolescents aged 15–19 y. The DNA damage response (DDR) is significantly enhanced in osteosarcoma, impairing the effect of systemic chemotherapy. Targeting the DDR process was considered a feasible strategy benefitting osteosarcoma patients. However, the clinical application of DDR inhibitors is not impressive because of their side effects. Chinese herbal medicines with high anti-tumor effects and low toxicity in the human body have gradually gained attention. 2-Hydroxy-3-methylanthraquinone (HMA), a Chinese medicine monomer found in the extract of Oldenlandia diffusa, exerts significant inhibitory effects on various tumors. However, its anti-osteosarcoma effects and defined molecular mechanisms have not been reported. Methods After HMA treatment, the proliferation and metastasis capacity of osteosarcoma cells was detected by CCK-8, colony formation, transwell assays and Annexin V-fluorescein isothiocyanate/propidium iodide staining. RNA-sequence, plasmid infection, RNA interference, Western blotting and immunofluorescence assay were used to investigate the molecular mechanism and effects of HMA inhibiting osteosarcoma. Rescue assay and CHIP assay was used to further verified the relationship between MYC, CHK1 and RAD51. Results HMA regulate MYC to inhibit osteosarcoma proliferation and DNA damage repair through PI3K/AKT signaling pathway. The results of RNA-seq, IHC, Western boltting etc. showed relationship between MYC, CHK1 and RAD51. Rescue assay and CHIP assay further verified HMA can impair homologous recombination repair through the MYC-CHK1-RAD51 pathway. Conclusion HMA significantly inhibits osteosarcoma proliferation and homologous recombination repair through the MYC-CHK1-RAD51 pathway, which is mediated by the PI3K-AKT signaling pathway. This study investigated the exact mechanism of the anti-osteosarcoma effect of HMA and provided a potential feasible strategy for the clinical treatment of human osteosarcoma. Graphical Abstract

Heritable loss‐of‐function mutations in genes encoding key regulators of DNA repair and genome stability can result in degenerative progeroid and/or cancer predisposition syndromes; however, such mutations have never been found to affect the Chk1 protein kinase, despite its central role in DNA damage signalling and checkpoint activation. Remarkably, two recent reports now demonstrate that heritable, gain‐of‐function mutations within the Chk1 C‐terminal regulatory domain can cause female infertility in humans. In vitro, oocytes from individuals heterozygous for such mutant Chk1 alleles fail to undergo the first mitotic division after fertilization owing to arrest in G2 phase of the cell cycle. This arrest results from inhibition of the master regulator of mitosis, the cyclin‐dependent kinase CDK1, through the same molecular mechanisms that are engaged by activated Chk1 to impose G2 checkpoint arrest in somatic cells bearing DNA damage. Remarkably, the failure of this first zygotic division in heterozygotes in vitro can be rescued through treatment with selective Chk1 inhibitor drugs, allowing development of apparently normal blastocysts and offering hope that a pharmacological solution to this cause of infertility may be possible. [ABSTRACT FROM AUTHOR]

Endocytosis has been shown to play an important role in cancer proliferation and metastasis. Recent studies have accumulated evidence that endocytosis inhibitors suppress in vitro and in vivo proliferation and migration. In addition, endocytosis inhibition has been shown to induce apoptosis, but its mechanism remains largely unclear. In this study, we found that the endocytosis inhibitor dynasore causes a cell viability reduction in multiple cancer cell lines, especially in hematopoietic cancers. Dynasore induced massive apoptosis and an S-phase progression delay. In addition, dynasore activated the ATR-Chk1 DNA damage response, which suggests a single-stranded DNA exposure induced by DNA replication stress. Furthermore, an ATR inhibitor sensitized the dynasore-induced apoptosis. These findings suggest that endocytosis inhibitors may have an ability to suppress DNA replication, a common mechanism of genotoxic chemotherapies targeting cancer, and that the anti-cancer effects of endocytosis inhibitors may be sensitized by DNA damage response inhibitors. • Anti-cancer activity of the dynamin inhibitor dynasore was investigated using eight human cancer cell lines. • Dynasore suppressed proliferation of hematopoietic cells more strongly than epithelial cells. • Dynasore induced apoptosis and the ATR-Chk1 DNA damage response pathway in Jurkat cells. • The ATR inhibitor VE-822 enhanced dynasore-induced apoptosis in Jurkat cells. [ABSTRACT FROM AUTHOR]

Simple Summary: The communication between the DNA damage response pathway and the tumor microenvironment of hepatocellular carcinoma (HCC) has attracted more attention. In this study, high CHK1 expression in HCC tissue was associated with advanced tumor stage and poor prognosis in patients. CHK1 inhibition and cisplatin induced cellular apoptosis via DNA damage in human HCC cells. CHK1 directly bound IRF1 and exerted a proteolytic effect in HCC cells induced by DNA damage. DNA damage induced MICA expression via IRF1 at the transcriptional level in HCC cells. MICA expression was positively correlated with NK cells and CD8+T cell infiltration in human HCC. This study provided new insight into the molecular mechanisms regulating HCC signaling with cross-talk between the IRF1 and CHK1 pathways. Background: CHK1 is considered a key cell cycle checkpoint kinase in DNA damage response (DDR) pathway to communicate with several signaling pathways involved in the tumor microenvironment (TME) in numerous cancers. However, the mechanism of CHK1 signaling regulating TME in hepatocellular carcinoma (HCC) remains unclear. Methods: CHK1 expression in HCC tissue was determined by IHC staining assay. DNA damage and apoptosis in HCC cells induced by cisplatin or CHK1 inhibition were detected by WB and flow cytometry. The interaction of CHK1 and IRF1 was analyzed by single-cell RNA-sequence, WB, and immunoprecipitation assay. The mechanism of IRF1 regulating MICA was investigated by ChIP-qPCR. Results: CHK1 expression is upregulated in human HCC tumors compared to the background liver. High CHK1 mRNA level predicts advanced tumor stage and worse prognosis. Cisplatin and CHK1 inhibition augment cellular DNA damage and apoptosis. Overexpressed CHK1 suppresses IRF1 expression through proteolysis. Furthermore, single-cell RNA-sequence analyses confirmed that MICA expression positively correlated with IRF1 in HCC cells. Immunoprecipitation assay showed the binding between CHK1 and IRF1. Cisplatin and CHK1 inhibition upregulate MICA expression through IRF1-mediated transcriptional effects. A novel specific cis-acting IRF response element was identified at -1756 bp in the MICA promoter region that bound IRF1 to induce MICA gene transcription. MICA may increase NK cell and CD8+T cell infiltration in HCC. Conclusions: DNA damage regulates the interaction of CHK1 and IRF1 to activate anti-tumor immunity via the IRF1-MICA pathway in HCC. [ABSTRACT FROM AUTHOR]

Simple Summary: DNA damage response (DDR)-related proteins contribute to tumorigenesis, tumor progression, and drug resistance. Pharmacological inhibition of DDR-related proteins, such as checkpoint kinase 1 (Chk1), exerts antitumor effects but has serious side effects. Therefore, we attempted to specifically kill cancer cells with low concentrations of drugs by simultaneously inhibiting two DDR-related proteins. In this study, we demonstrated that the combined treatment with ataxia telangiectasia-mutated serine/threonine kinase (ATM) inhibitor (ATMi) and Chk1 inhibitor (Chk1i) exerts synergistic antitumor effects and induces cancer-specific synthetic lethality at low doses. The ATMi and Chk1i combination synergistically promotes CDK1 activation, resulting in the induction of apoptosis with enhanced cell cycle progression in cancer cells. Considering that the combined treatment exerts antitumor effects at one-tenth the concentrations described in previous reports, with high antitumor efficacy and few side effects it could be an effective treatment approach. Genetic abnormalities induce the DNA damage response (DDR), which enables DNA repair at cell cycle checkpoints. Although the DDR is thought to function in preventing the onset and progression of cancer, DDR-related proteins are also thought to contribute to tumorigenesis, tumor progression, and drug resistance by preventing irreparable genomic abnormalities from inducing cell death. In the present study, the combination of ataxia telangiectasia-mutated serine/threonine kinase (ATM) and checkpoint kinase 1 (Chk1) inhibition exhibited synergistic antitumor effects and induced synergistic lethality in colorectal cancer cells at a low dose. The ATM and Chk1 inhibitors synergistically promoted the activation of cyclin-dependent kinase 1 by decreasing the phosphorylation levels of T14 and Y15. Furthermore, the combined treatment increased the number of sub-G1-stage cells, phospho-histone H2A.X-positive cells, and TdT-mediated dUTP nick-end labeling-positive cells among colon cancer cells, suggesting that the therapy induces apoptosis. Finally, the combined treatment exhibited a robust antitumor activity in syngeneic tumor model mice. These findings should contribute to the development of new treatments for colorectal cancer that directly exploit the genomic instability of cancer cells. [ABSTRACT FROM AUTHOR]

Replication stress (RS) is a characteristic state of cancer cells as they tend to exchange precision of replication for fast proliferation and increased genomic instability. To overcome the consequences of improper replication control, malignant cells frequently inactivate parts of their DNA damage response (DDR) pathways (the ATM-CHK2-p53 pathway), while relying on other pathways which help to maintain replication fork stability (ATR-CHK1). This creates a dependency on the remaining DDR pathways, vulnerability to further destabilization of replication and synthetic lethality of DDR inhibitors with common oncogenic alterations such as mutations of TP53, RB1, ATM, amplifications of MYC, CCNE1 and others. The response to RS is normally limited by coordination of cell cycle, transcription and replication. Inhibition of WEE1 and PKMYT1 kinases, which prevent unscheduled mitosis entry, leads to fragility of under-replicated sites. Recent evidence also shows that inhibition of Cyclin-dependent kinases (CDKs), such as CDK4/6, CDK2, CDK8/19 and CDK12/13 can contribute to RS through disruption of DNA repair and replication control. Here, we review the main causes of RS in cancers as well as main therapeutic targets—ATR, CHK1, PARP and their inhibitors.

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. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma is a malignant bone tumor that usually affects adolescents aged 15–19 y. The DNA damage response (DDR) is significantly enhanced in osteosarcoma, impairing the effect of systemic chemotherapy. Targeting the DDR process was considered a feasible strategy benefitting osteosarcoma patients. However, the clinical application of DDR inhibitors is not impressive because of their side effects. Chinese herbal medicines with high anti-tumor effects and low toxicity in the human body have gradually gained attention. 2-Hydroxy-3-methylanthraquinone (HMA), a Chinese medicine monomer found in the extract of Oldenlandia diffusa, exerts significant inhibitory effects on various tumors. However, its anti-osteosarcoma effects and defined molecular mechanisms have not been reported. Methods: After HMA treatment, the proliferation and metastasis capacity of osteosarcoma cells was detected by CCK-8, colony formation, transwell assays and Annexin V-fluorescein isothiocyanate/propidium iodide staining. RNA-sequence, plasmid infection, RNA interference, Western blotting and immunofluorescence assay were used to investigate the molecular mechanism and effects of HMA inhibiting osteosarcoma. Rescue assay and CHIP assay was used to further verified the relationship between MYC, CHK1 and RAD51. Results: HMA regulate MYC to inhibit osteosarcoma proliferation and DNA damage repair through PI3K/AKT signaling pathway. The results of RNA-seq, IHC, Western boltting etc. showed relationship between MYC, CHK1 and RAD51. Rescue assay and CHIP assay further verified HMA can impair homologous recombination repair through the MYC-CHK1-RAD51 pathway. Conclusion: HMA significantly inhibits osteosarcoma proliferation and homologous recombination repair through the MYC-CHK1-RAD51 pathway, which is mediated by the PI3K-AKT signaling pathway. This study investigated the exact mechanism of the anti-osteosarcoma effect of HMA and provided a potential feasible strategy for the clinical treatment of human osteosarcoma. [ABSTRACT FROM AUTHOR]

Check point kinase 1 (Chk1) is an essential protein in G2 phase checkpoint arrest, which cancer cells need to sustain the cell cycle and prevent cell death. Chk1 inhibitors have been shown to eliminate the S and G2 checkpoints and change the DNA repair pathway, resulting in immature mitotic progression, mitotic catastrophe, and cell death. Normal cells remain in the G1 phase to repair DNA damage as a result of p53 and are less affected by the deletion of the S and G2 checkpoints. Due of its function in this research we have tried to target CHK1 to identify potent CHK1 inhibitors by employing computer aided drug design. Million Molecules Database, Natural Product Database, NCI Database has been screened and three molecules has been identified by structure-based virtual screening followed by filtering for various drug likeness, ADME, toxicity, Molecular docking. Our research work resulted in lead molecules that have shown strong binding affinity with effective ADME properties, low toxicity, and high stability. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer is more aggressive than other types of breast cancer. Protein kinase R (PKR), which is activated by dsRNA, is known to play a role in doxorubicin-mediated apoptosis; however, its role in DNA damage-mediated apoptosis is not well understood. In this study, we investigated the roles of PKR and its downstream players in doxorubicin-treated HCC1143 triple-negative breast cancer cells. Doxorubicin treatment induces DNA damage and apoptosis. Interestingly, doxorubicin treatment induced the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) via PKR, whereas the inhibition of PKR with inhibitor C16 reduced eIF2α phosphorylation. Under these conditions, doxorubicin-mediated DNA fragmentation, cell death, and poly(ADP ribose) polymerase and caspase 7 levels were recovered. In addition, phosphorylation of checkpoint kinase 1 (CHK1), which is known to be involved in doxorubicin-mediated DNA damage, was increased by doxorubicin treatment, but blocked by PKR inhibition. Protein translation was downregulated by doxorubicin treatment and upregulated by blocking PKR phosphorylation. These results suggest that PKR activation induces apoptosis by increasing the phosphorylation of eIF2α and CHK1 and decreasing the global protein translation in doxorubicin-treated HCC1143 triple-negative breast cancer cells. [ABSTRACT FROM AUTHOR]

BRCA1 is a well-known breast cancer risk gene, involved in DNA damage repair via homologous recombination (HR) and replication fork protection. Therapy resistance was linked to loss and amplification of the BRCA1 gene causing inferior survival of breast cancer patients. Most studies have focused on the analysis of complete loss or mutations in functional domains of BRCA1. How mutations in non-functional domains contribute to resistance mechanisms remains elusive and was the focus of this study. Therefore, clones of the breast cancer cell line MCF7 with indels in BRCA1 exon 9 and 14 were generated using CRISPR/Cas9. Clones with successful introduced BRCA1 mutations were evaluated regarding their capacity to perform HR, how they handle DNA replication stress (RS), and the consequences on the sensitivity to MMC, PARP1 inhibition, and ionizing radiation. Unexpectedly, BRCA1 mutations resulted in both increased sensitivity and resistance to exogenous DNA damage, despite a reduction of HR capacity in all clones. Resistance was associated with improved DNA double-strand break repair and reduction in replication stress (RS). Lower RS was accompanied by increased activation and interaction of proteins essential for the S phase-specific DNA damage response consisting of HR proteins, FANCD2, and CHK1. [ABSTRACT FROM AUTHOR]

Abstract Background Challenges exist in the clinical treatment of luminal estrogen receptor α (ERα)-positive breast cancers (BCs) both to prevent resistance to endocrine therapy (ET) and to treat ET-resistant metastatic BCs (MBC). Therefore, we evaluated if kinases could be new targets for the treatment of luminal primary and MBCs. Methods ~ 170 kinase inhibitors were applied to MCF-7 cells either with adaptative or genetic resistance to ET drugs and both ERα levels and cell proliferation were measured. Robust-Z-score calculation identified AZD7762 (CHK1/CHK2 inhibitor) as a positive hit. Subsequently, Kaplan–Meier analyses of CHK1 and CHK2 impact on ERα-positive BC patients relapse-free-survival (RFS), bioinformatic evaluations of CHK1 and CHK2 expression and activation status as a function of ERα activation status as well as drug sensitivity studies in ERα-positive BC cell lines, validation of the impact of the ATR:CHK1 and ATM:CHK2 pathways on the control of ERα stability and BC cell proliferation via inhibitor- and siRNA-based approaches, identification of the molecular mechanism required for inhibitor-dependent ERα degradation in BC and the impact of CHK1 and CHK2 inhibition on the 17β-estradiol (E2):ERα signaling, synergy proliferation studies between ET-drugs and clinically relevant CHK1 inhibitors in different luminal BC cell lines, were performed. Results A reduced CHK1 expression correlates with a longer RFS in women with ERα-positive BCs. Interestingly, women carrying luminal A BC display an extended RFS when expressing low CHK1 levels. Accordingly, CHK1 and ERα activations are correlated in ERα-positive BC cell lines, and the ATR:CHK1 pathway controls ERα stability and cell proliferation in luminal A BC cells. Mechanistically, the generation of DNA replication stress rather than DNA damage induced by ATR:CHK1 pathway inhibition is a prerequisite for ERα degradation. Furthermore, CHK1 inhibition interferes with E2:ERα signaling to cell proliferation, and drugs approved for clinical treatment of primary and MBC (4OH-tamoxifen and the CDK4/CDK6 inhibitors abemaciclib and palbociclib) exert synergic effects with the CHK1 inhibitors in clinical trials for the treatment of solid tumors (AZD7762, MK8776, prexasertib) in preventing the proliferation of cells modeling primary and MBC. Conclusions CHK1 could be considered as an appealing novel pharmacological target for the treatment of luminal primary and MBCs.

[Display omitted] • Two series of imidazolones and imidazothienopyrimidines were synthesized. • All compounds were screened in the NCI 60 cancer cell lines. • Compound 4f displayed potent dual inhibition of both Chk1 and Chk2. • Compound 4f arrested the cell cycle of EKVX at S phase by inducing apoptosis. • The in silico profiles of the most active analogs have been carried. Applying various drug design strategies including ring variation, substituents variation, and ring fusion, two series of 2-(alkylthio)-5-(arylidene/heteroarylidene)imidazolones and imidazo[1,2- a ]thieno[2,3- d ]pyrimidines were designed and prepared as dual potential Chk1 and Chk2 inhibitors. The newly synthesized hybrids were screened in NCI 60 cell line panel where the most active derivatives 4b, d-f , and 6a were further estimated for their five dose antiproliferative activity against the most sensitive tumor cells including breast MCF-7 and MDA-MB-468 and non-small cell lung cancer EKVX as well as normal WI-38 cell. Noticeably, increasing the carbon chain attached to thiol moiety at C-2 of imidazolone scaffold elevated the cytotoxic activity. Hence, compounds 4e and 4f , containing S-butyl fragment, exhibited the most antiproliferative activity against the tested cells where 4f showed extremely potent selectivity toward them. As well, compound 6a , containing imidazothienopyrimidine core, exerted significant cytotoxic activity and selectivity toward the examined cells. The mechanistic investigation of the most active cytotoxic analogs was achieved through the evaluation of their inhibitory activity against Chk1 and Chk2. Results revealed that 4f displayed potent dual inhibition of both Chk1 and Chk2 with IC 50 equal 0.137 and 0.25 μM, respectively. It also promoted its antiproliferative and Chk suppression activity via EKVX cell cycle arrest at S phase through stimulating the apoptotic approach. The apoptosis induction was also emphasized by elevating the expression of Caspase-3 and Bax, that are accompanied by Bcl-2 diminution. The in silico molecular docking and ADMET profiles of the most active analogs have been carried out to evaluate their potential as significant anticancer drug candidates. [ABSTRACT FROM AUTHOR]

[Display omitted] Apelin-13, a type of active peptide, can alleviate lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the specific mechanism is unclear. Cell cycle checkpoint kinase 1 (Chk1) plays an important role in DNA damage. Here, we investigated the regulatory effect of Apelin on Chk1 in ALI. Chk1-knockout and −overexpression mice were used to explore the role of Chk1 in LPS-induced ALI mice treated with or without Apelin-13. In addition, A549 cells were also treated with LPS to establish a cell model. Chk1 knockdown inhibited the destruction of alveolar structure, the damage of lung epithelial barrier function, and DNA damage in the ALI mouse model. Conversely, Chk1 overexpression had the opposite effect. Furthermore, Apelin-13 reduced Chk1 expression and DNA damage to improve the impaired lung epithelial barrier function in the ALI model. However, the high expression of Chk1 attenuated the protective effect of Apelin-13 on ALI. Notably, Apelin-13 promoted Chk1 degradation through autophagy to regulate DNA damage in LPS-treated A549 cells. In summary, Apelin-13 regulates the expression of Chk1 by promoting autophagy, thereby inhibiting epithelial DNA damage and repairing epithelial barrier function. [ABSTRACT FROM AUTHOR]

Aim: The present study was designed to evaluate the role of DNA damage response pathway genes and heat-shock proteins in head and neck cancer (HNC) risk. Methods: For this purpose, two study cohorts were used. Cohort 1 (blood samples of 250 HNC patients and 250 controls) was used for polymorphism screening of selected genes using tetra-primer amplification refractory mutation system-polymerase chain (Tetra-ARMS PCR). Cohort 2 (200 HNC tumors and adjacent controls) was used for expression analysis, using quantitative PCR. Results: Analysis showed that mutant allele frequency of selected polymorphisms was found associated with increased HNC risk. Expression analysis showed the significant deregulation of selected genes in patients. Conclusion: The present study showed that selected genes (CHK1, CHK2, HSP70 and HSP90) can act as good diagnostic/prognostic markers in HNC. The present study is designed to identify the selected genes of DNA damage response pathway and heat-shock proteins as diagnostic/prognostic markers of head and neck cancer (HNC). To do this, DNA was isolated from blood samples and RNA isolated from the tissue samples of HNC patients. The mutation and expression level of selected genes was tested, and selected genes showed good diagnostic/prognostic values for HNC patients. [ABSTRACT FROM AUTHOR]

Purpose: The aim of the current study was to assess the prognostic value of the Chk1 gene in the DNA damage response pathway in gastric cancer (GC). Methods: Expression levels of the Chk1 were measured in 220 GC tumor tissues and adjacent healthy/noncancerous tissues using real-time PCR and immunohistochemical staining. Genomic instability in GC patients was measured using the long-run real-time PCR technique for DNA-damage quantification assay and comet assay. Results: Significantly downregulated expression of Chk1 was observed at the mRNA level (p < 0.0001) and protein level (p < 0.0001). Significantly increased frequency of lesions/10 kb and comets was observed in tumor tissues compared with control tissues. Conclusion: The data suggest that downregulated expression of Chk1 and positive Heliobacter pylori infection status may have prognostic significance in GC. [ABSTRACT FROM AUTHOR]

Background: Both phospholipid synthesis and the detection of DNA damage are coupled to cell cycle progression, yet whether these two aspects crosstalk to each other remains unassessed. We postulate here that shortage of phospholipids, which negatively affects proliferation, may reduce the need for checkpoint activation in response to DNA damage. Results: To test this hypothesis, we explore here the DNA Damage Response activation in response to seven different genotoxins, in three distinct cell types, and manipulate phospholipid synthesis both pharmacologically and genetically. This allows us to point at the DNA damage response kinase ATR as responsible for the coordination between phospholipid levels and DNA damage sensing. Conclusions and Significance: ATR could combine its ability to sense DNA damage and phospholipid profiles in order to finetune the response to DNA lesions depending on metabolic cues. Further, our analysis reveals the functional significance of this crosstalk to keep genome homeostasis. [ABSTRACT FROM AUTHOR]

Purpose: The aim of the current study was to assess the prognostic value of the Chk1 gene in the DNA damage response pathway in gastric cancer (GC). Methods: Expression levels of the Chk1 were measured in 220 GC tumor tissues and adjacent healthy/noncancerous tissues using real-time PCR and immunohistochemical staining. Genomic instability in GC patients was measured using the long-run real-time PCR technique for DNA-damage quantification assay and comet assay. Results: Significantly downregulated expression of Chk1 was observed at the mRNA level (p < 0.0001) and protein level (p < 0.0001). Significantly increased frequency of lesions/10 kb and comets was observed in tumor tissues compared with control tissues. Conclusion: The data suggest that downregulated expression of Chk1 and positive Heliobacter pylori infection status may have prognostic significance in GC. [ABSTRACT FROM AUTHOR]

Background: Aronia berry extracts (ABE) have recently been reported to possess significant anti-cancer effects in various malignancies, including colorectal cancer (CRC), due to their high polyphenolic content. However, the molecular mechanism(s) underlying the anti-cancer effects of ABE in CRC remain unclear, which is important to consider when considering their use as complementary medicine approaches in cancer., Methods: We performed genome-wide transcriptomic profiling and pathway enrichment analysis to identify specific growth signaling pathways associated with ABE treatment in CRC cells. In addition, a series of systematic and comprehensive cell culture studies were performed to investigate the anti-cancer effects of ABE in SW480 and HCT116 CRC cell lines. Subsequently, these findings were validated in patient-derived 3D organoids (PDOs) models., Results: Transcriptomic profiling analysis identified p53 signaling as one of the key enriched pathways mediating the anti-cancer activity of ABE. Analysis of public datasets revealed that Chk1, a key regulator of p53, was one of the critical targets of ABE in CRC. Chk1 and p53 activation was shown to be downregulated with ABE treatment, leading to the induction of cell cycle arrest (p = 0.003-0.014) and enhanced DNA damage (p = 0.015-0.026). Furthermore, these findings were validated in PDOs, where the ABE treatment resulted in significantly fewer and smaller PDOs in a concentration-dependent manner (p = 0.045 - <0.001)., Conclusions: We firstly provide evidence for the role of the p53 signaling pathway as a mediator of the anti-cancer activity of ABE, which provides a rationale for its use as a safe and effective integrative medicine approach in CRC., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to report., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The relationship between O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and mitosis is intertwined. Besides the numerous mitotic OGT substrates that have been identified, OGT itself is also a target of the mitotic machinery. Previously, our investigations have shown that Checkpoint kinase 1 (Chk1) phosphorylates OGT at Ser-20 to increase OGT levels during cytokinesis, suggesting that OGT levels oscillate as mitosis progresses. Herein we studied its underlying mechanism. We set out from an R17C mutation of OGT, which is a uterine carcinoma mutation in The Cancer Genome Atlas. We found that R17C abolishes the S20 phosphorylation of OGT, as it lies in the Chk1 phosphorylating consensus motif. Consistent with our previous report that pSer-20 is essential for OGT level increases during cytokinesis, we further demonstrate that the R17C mutation renders OGT less stable, decreases vimentin phosphorylation levels and results in cytokinesis defects. Based on bioinformatic predictions, pSer-20 renders OGT more likely to interact with 14-3-3 proteins, the phospho-binding signal adaptor/scaffold protein family. By screening the seven isoforms of 14-3-3 family, we show that 14-3-3ε specifically associates with Ser-20-phosphorylated OGT. Moreover, we studied the R17C and S20A mutations in xenograft models and demonstrated that they both inhibit uterine carcinoma compared to wild-type OGT, probably due to less cellular reproduction. Our work is a sequel of our previous report on pS20 of OGT and is in line with the notion that OGT is intricately regulated by the mitotic network., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12 to 15 hours, after which cells "adapt" to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well-understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2 ATRIP , Rad9, Rad24, or Rad53 CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and to maintain the arrest. Dun1 is required for establishment, but not maintenance of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with 2 persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hours these proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2's binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest requires overlapping but different sets of factors., Competing Interests: Competing Interest Statement The authors declare no competing interests.

The ATR kinase protects cells against DNA damage and replication stress and represents a promising anti-cancer drug target. The ATR inhibitors (ATRi) berzosertib and gartisertib are both in clinical trials for the treatment of advanced solid tumors as monotherapy or in combination with genotoxic agents. We carried out quantitative phospho-proteomic screening for ATR biomarkers that are highly sensitive to berzosertib and gartisertib, using an optimized mass spectrometry pipeline. Screening identified a range of novel ATR-dependent phosphorylation events, which were grouped into three broad classes: (i) targets whose phosphorylation is highly sensitive to ATRi and which could be the next generation of ATR biomarkers; (ii) proteins with known genome maintenance roles not previously known to be regulated by ATR; (iii) novel targets whose cellular roles are unclear. Class iii targets represent candidate DNA damage response proteins and, with this in mind, proteins in this class were subjected to secondary screening for recruitment to DNA damage sites. We show that one of the proteins recruited, SCAF1, interacts with RNAPII in a phospho-dependent manner and recruitment requires PARP activity and interaction with RNAPII. We also show that SCAF1 deficiency partly rescues RAD51 loading in cells lacking the BRCA1 tumor suppressor. Taken together these data reveal potential new ATR biomarkers and new genome maintenance factors., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We have recently reported that in G2-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the G2-checkpoint epistatically, with ATR at the output-node, interfacing with the cell cycle through Chk1. However, although inhibition of ATR nearly completely abrogated the checkpoint, inhibition of Chk1 using UCN-01 generated only partial responses. This suggested that additional kinases downstream of ATR were involved in the transmission of the signal to the cell cycle engine. Additionally, the broad spectrum of kinases inhibited by UCN-01 pointed to uncertainties in the interpretation that warranted further investigations. Here, we show that more specific Chk1 inhibitors exert an even weaker effect on G2-checkpoint, as compared to ATR inhibitors and UCN-01, and identify the MAPK p38α and its downstream target MK2 as checkpoint effectors operating as backup to Chk1. These observations further expand the spectrum of p38/MK2 signaling to G2-checkpoint activation, extend similar studies in cells exposed to other DNA damaging agents and consolidate a role of p38/MK2 as a backup kinase module, adding to similar backup functions exerted in p53 deficient cells. The results extend the spectrum of actionable strategies and targets in current efforts to enhance the radiosensitivity in tumor cells.