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DNA damage and repair have been shown to be associated with carotid artery restenosis and atherosclerosis. The proliferation and migration of vascular smooth muscle cells (VSMCs) is the main cause of artery stenosis. This study aims to define the relationship between DNA damage and VSMCs proliferation. A rat carotid artery injury model was established, and human and rat VSMCs cultured in vitro. H 2 O 2 was used to induce DNA damage in vitro. The selected CHK1 inhibitor, LY2603618, was used to inhibit CHK1 phosphorylation both in vivo and in vitro. γH2AX, αSMA and phosphorylated CHK1 were detected both in rat carotid artery and cultured VSMCs from different groups. Hyperplasia ratio of rat carotid artery intimal was measured. DNA double-strand breaks occur in the rat carotid artery after injury. DNA damage induces CHK1 phosphorylation and down-regulates αSMA expression in VSMCs both in vitro and in vivo. The inhibition of CHK1 phosphorylation rescues αSMA expression in VSMCs both in vitro and in vivo , and rat carotid intimal hyperplasia after injury was suppressed. Our data demonstrated that phosphorylation of CHK1 under DNA damage stress modulates VSMCs phenotypic switching. CHK1 inhibition may be a potential therapeutic strategy for intima hyperplasia treatment. Image 1 • DNA damage was observed in carotid artery tissues from a rat carotid artery injury model. • DNA damage activates CHK1 and leads to vascular smooth muscle cells (VSMCs) phenotypic switching both in vitro and in vivo. • The phosphorylation of CHK1 at Ser 345 regulates VSMCs phenotype switching. • CHK1 inhibition suppressed the phenotypic switching of VSMCs both in vitro and in vivo. • CHK1 inhibition reduced the intimal hyperplasia in carotid artery after balloon injury. [ABSTRACT FROM AUTHOR]

Summary: Oncoproteins such as the BRAFV600E kinase endow cancer cells with malignant properties, but they also create unique vulnerabilities. Targeting of BRAFV600E-driven cytoplasmic signaling networks has proved ineffective, as patients regularly relapse with reactivation of the targeted pathways. We identify the nuclear protein SFPQ to be synthetically lethal with BRAFV600E in a loss-of-function shRNA screen. SFPQ depletion decreases proliferation and specifically induces S-phase arrest and apoptosis in BRAFV600E-driven colorectal and melanoma cells. Mechanistically, SFPQ loss in BRAF-mutant cancer cells triggers the Chk1-dependent replication checkpoint, results in decreased numbers and reduced activities of replication factories, and increases collision between replication and transcription. We find that BRAFV600E-mutant cancer cells and organoids are sensitive to combinations of Chk1 inhibitors and chemically induced replication stress, pointing toward future therapeutic approaches exploiting nuclear vulnerabilities induced by BRAFV600E.

Ovarian cancer is the most lethal gynecological malignancy. Abnormal homologous recombination repair, high level of reactive oxygen species (ROS) and upregulation of antioxidant genes are characteristic features of ovarian cancer. However, the molecular mechanisms governing the redox homeostasis in ovarian cancer cells remain to be fully elucidated. We here demonstrated a critical role of RAD51, a protein essential for homologous recombination, in the maintenance of redox homeostasis. We found that RAD51 is overexpressed in high grade serous ovarian cancer and is associated with poor prognosis. Depletion or inhibition of RAD51 results in G2/M arrest, increased production of reactive oxygen species and accumulation of oxidative DNA damage. Importantly, antioxidant N-acetylcysteine (NAC) significantly attenuated the induction of DNA damage and the perturbation of proliferation caused by RAD51 depletion. We further demonstrated that RAD51 inhibition or depletion led to elevated production of mitochondrial superoxide and increased accumulation of mitochondria. Moreover, CHK1 activation is required for the G2/M arrest and the generation of mitochondrial stress in response to RAD51 depletion. Together, our results indicate that nuclear DNA damage caused by RAD51 depletion may trigger mitochondria-originated redox dysregulation. Our findings suggest that a vicious cycle of nuclear DNA damage, mitochondrial accumulation and oxidative stress may contribute to the tumor-suppressive effects of RAD51 depletion or inhibition.

ABSTRACT The covalently closed circular (CCC) DNA of hepatitis B virus (HBV) functions as the only viral transcriptional template capable of producing all viral RNA species and is essential to initiate and sustain viral replication. CCC DNA is converted from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. As RC DNA mimics damaged cellular DNA, the host cell DNA damage repair (DDR) system is thought to be responsible for HBV CCC DNA formation. The potential role of two major cellular DDR pathways, the ataxia telangiectasia mutated (ATM) pathway and the ATM and Rad3-related (ATR) pathway, in HBV CCC DNA formation was thus investigated. Inhibition, or expression knockdown, of ATR and its downstream signaling factor CHK1, but not of ATM, decreased CCC DNA formation during de novo HBV infection, as well as intracellular CCC DNA amplification, when RC DNA from extracellular virions and intracellular nucleocapsids, respectively, is converted to CCC DNA. Furthermore, a novel RC DNA processing product with 5′ truncated minus strands was detected when the ATR-CHK1 pathway was inhibited, further indicating that this pathway controls RC DNA processing during its conversion to CCC DNA. These results provide new insights into how host cells recognize and process HBV RC DNA in order to produce CCC DNA and have implications for potential means to block CCC DNA production. IMPORTANCE Hepatitis B virus (HBV) chronically infects hundreds of millions of people and remains a major cause of viral hepatitis, cirrhosis, and liver cancer. HBV persistence is sustained by a viral nuclear episome that directs all viral gene expression needed to support viral replication. The episome is converted from an incomplete DNA precursor in viral particles in an ill-understood process. We report here that the incomplete DNA precursor is recognized by the host cell in a way similar to the sensing of damaged cellular DNA for subsequent repair to form the nuclear episome. Intense efforts are ongoing to develop novel antiviral strategies to eliminate CCC DNA so as to cure chronic HBV infection. Our results here provide novel insights into, and suggest novel ways of perturbing, the process of episome formation. Furthermore, our results inform mechanisms of cellular DNA damage recognition and repair, processes essential for normal cell growth.

The DNA damage response enables cells to cope with various stresses that threaten genomic integrity. A critical component of this response is the serine/threonine kinase CHK1 which is encoded by the CHEK1 gene. Originally identified as a regulator of the G2/M checkpoint, CHK1 has since been shown to play important roles in DNA replication, mitotic progression, DNA repair, and overall cell cycle regulation. However, the potential of CHK1 as a cancer therapy has not been realized clinically. Herein we expound our current understanding of the principal roles of CHK1 and highlight different avenues for CHK1 targeting in cancer therapy. [ABSTRACT FROM AUTHOR]

Rad3 is the orthologue of ATR and the sensor kinase of the DNA replication checkpoint in Schizosaccharomyces pombe. Under replication stress, it initiates checkpoint signaling at the forks necessary for maintaining genome stability and cell survival. To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome, looking for mutants defective in response to the replication stress induced by hydroxyurea. In addition to the previously reported mutant with a C-to-Y change at position 307 encoded by tel2 (tel2-C307Y mutant) (Y.-J. Xu, S. Khan, A. C. Didier, M. Wozniak, et al., Mol Cell Biol 39:e00175-19, 2019, https://doi .org/10.1128/MCB.00175-19), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations, except for a start codon mutation, are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint. In support of this notion, integration of two helicase-inactive mutations or deletion of rqh1 generated a similar Rad3 signaling defect, and heterologous expression of human RECQ1, BLM, and RECQ4 restored the Rad3 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication checkpoint function of Rqh1 is highly conserved, and mutations in the helicase domain of these human enzymes may cause the checkpoint defect and contribute to the cancer predisposition syndromes. [ABSTRACT FROM AUTHOR]

Summary: Purpose The primary objective was to determine the recommended Phase 2 dose (RP2D) of checkpoint kinase 1 inhibitor, prexasertib, in combination with the p38 mitogen-activated protein kinase inhibitor, ralimetinib, which may be safely administered to patients with advanced cancer. Methods This Phase 1, nonrandomized, open-label, dose-escalation study of prexasertib+ralimetinib included patients with advanced and/or metastatic cancer, followed by a planned cohort expansion in patients with colorectal or non-small-cell lung cancer with KRAS and/or BRAF mutations. Intravenous prexasertib was administered at 60 mg/m2 (days 1 and 15 of a 28-day cycle), together with oral ralimetinib every 12 h (days 1 to 14 at 100 mg [Cohort 1, n = 3] or 200 mg [Cohort 2, n = 6]). Dose escalations for each agent were planned using a model-based 3 + 3 escalation paradigm. Safety was assessed using Common Terminology Criteria for Adverse Events (CTCAE) v4.0X. Tumor response was determined by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Results Nine patients were treated; 3 experienced dose-limiting toxicities, all in Cohort 2, prohibiting further dose escalation. The most common ≥Grade 3 adverse event was neutrophil count decreased; other reported ≥Grade 3 hematological toxicities included febrile neutropenia and anemia. The pharmacokinetics of prexasertib+ralimetinib was comparable to the monotherapy population profile for each agent. One patient achieved a best overall response of stable disease (for 2 cycles); there were no complete/partial responses. Conclusions This study did not achieve its primary objective of establishing an RP2D of combination prexasertib + ralimetinib that could be safely administered to patients with advanced cancer. [ABSTRACT FROM AUTHOR]

In germ cells undergoing meiosis, the induction of double strand breaks (DSBs) is required for the generation of haploid gametes. Defects in the formation, detection, or recombinational repair of DSBs often result in defective chromosome segregation and aneuploidies. Central to the ability of meiotic cells to properly respond to DSBs are DNA damage response (DDR) pathways mediated by DNA damage sensor kinases. DDR signaling coordinates an extensive network of DDR effectors to induce cell cycle arrest and DNA repair, or trigger apoptosis if the damage is extensive. Despite their importance, the functions of DDR kinases and effector proteins during meiosis remain poorly understood and can often be distinct from their known mitotic roles. A key DDR kinase during meiosis is ataxia telangiectasia and Rad3‐related (ATR). ATR mediates key signaling events that control DSB repair, cell cycle progression, and meiotic silencing. These meiotic functions of ATR depend on upstream scaffolds and regulators, including the 9‐1‐1 complex and TOPBP1, and converge on many downstream effectors such as the checkpoint kinase CHK1. Here, we review the meiotic functions of the 9‐1‐1/TOPBP1/ATR/CHK1 signaling pathway during mammalian meiosis. [ABSTRACT FROM AUTHOR]

The most prevalent human carcinogen is sunlight-associated ultraviolet (UV), a physiologic dose of which generates thousands of DNA lesions per cell, mostly of two types: cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). It has not been possible, in living cells, to precisely characterize the respective contributions of these two lesion types to the signals that regulate cell cycle progression, DNA replication, and cell survival. Here we coupled multiparameter flow cytometry with lesion-specific photolyases that eliminate either CPDs or 6-4PPs and determined their respective contributions to DNA damage responses. Strikingly, only 6-4PP lesions activated the ATR-Chk1 DNA damage response pathway. Mechanistically, 6-4PPs, but not CPDs, impeded DNA replication across the genome as revealed by microfluidic-assisted replication track analysis. Furthermore, single-stranded DNA accumulated preferentially at 6-4PPs during DNA replication, indicating selective and prolonged replication blockage at 6-4PPs. These findings suggest that 6-4PPs, although eightfold fewer in number than CPDs, are the trigger for UV-induced DNA damage responses. [ABSTRACT FROM AUTHOR]

The DNA damage response recognizes DNA lesions and coordinates a cell cycle arrest with the repair of the damaged DNA, or removal of the affected cells to prevent the passage of genetic alterations to the next generation. The mitotic cell division, on the other hand, is a series of processes that aims to accurately segregate the genomic material from the maternal to the two daughter cells. Despite their great importance in safeguarding genomic integrity, the DNA damage response and the mitotic cell division were long viewed as unrelated processes, mainly because animal cells that are irradiated during mitosis continue cell division without repairing the broken chromosomes. However, recent studies have demonstrated that DNA damage proteins play an important role in mitotic cell division. This is performed through regulation of the onset of mitosis, mitotic spindle formation, correction of misattached kinetochore–microtubules, spindle checkpoint signaling, or completion of cytokinesis (abscission), in the absence of DNA damage. In this review, we summarize the roles of DNA damage proteins in unperturbed mitosis, analyze the molecular mechanisms involved, and discuss the potential implications of these findings in cancer therapy. [ABSTRACT FROM AUTHOR]

The checkpoint kinase, CHK1 plays an important role in repairing DNA damage induced by genotoxic agents for cancer treatment, which renders the cells resistant to such treatment modalities. Therefore, its inhibition appeared as a highly attractive strategy to re-sensitize cancer cells to the DNA damaging chemo and radiotherapies. In this study, a structure-based drug design approach was employed to identify potential ATP competitive CHK1 inhibitors. To this end, 3 crystal structures of CHK1 in complex with ATP competitive inhibitors were utilized to generate structure-based pharmacophore models that were subsequently used for virtual screening of commercial databases. Retrieved hits were filtered, then they were docked into the binding site of the enzyme and the free binding energies of the top-ranked docked hits were calculated. Based on the in silico results, 10 compounds were selected and their CHK1 inhibitory potential was evaluated in vitro. Noteworthy, in this exploratory study compounds 7 and 9 showed moderate but significant inhibition of the catalytic activity of the CHK1 enzyme. These two compounds were identified as promising hits worthy of further optimization towards designing better leads with druglike properties as potential competitive CHK1 inhibitors. [ABSTRACT FROM AUTHOR]

Ovarian cancer is one of the most lethal gynecologic malignancies reported throughout the world. The initial, standard-of-care, adjuvant chemotherapy in epithelial ovarian cancer is usually a platinum drug, such as cisplatin or carboplatin, combined with a taxane. However, despite surgical removal of the tumor and initial high response rates to first-line chemotherapy, around 80% of women will develop cancer recurrence. Effective strategies, including chemotherapy and new research models, are necessary to improve the prognosis. The replication stress response (RSR) is characteristic of the development of tumors, including ovarian cancer. Hence, RSR pathway and DNA repair proteins have emerged as a new area for anticancer drug development. Although clinical trials have shown poly (ADP-ribose) polymerase inhibitors (PARPi) response rates of around 40% in women who carry a mutation in the BRCA1/2 genes, PARPi is responsible for tumor suppression, but not for complete tumor regression. Recent reports suggest that cells with impaired homologous recombination (HR) activities due to mutations in TP53 gene or specific DNA repair proteins are specifically sensitive to ataxia telangiectasia and Rad3-related protein (ATR) inhibitors. Replication stress activates DNA repair checkpoint proteins (ATR, CHK1), which prevent further DNA damage. This review describes the use of DNA repair checkpoint inhibitors as single agents and strategies combining these inhibitors with DNA-damaging compounds for ovarian cancer therapy, as well as the new platforms used for optimizing ovarian cancer therapy. [ABSTRACT FROM AUTHOR]

DNA damage repair is a key factor in the maintenance of cell genome stability, plays an important role in the regulation of tumour evolution, and can affect the prognosis of cancer patients. This study aimed to detect the protein expression of the DNA damage repair protein P53 and its upstream and downstream regulators, CHK1, GADD45A, and MDM2, in oral squamous cell carcinoma (OSCC), in order to analyse the association between the expression of these proteins and overall survival, and to assess their prognostic implications for OSCC patients. The expression of the above proteins was detected by immunohistochemistry in 80 human OSCC tissue samples and in non‐cancerous tissue samples. Compared to that in the non‐cancerous tissue, the expression of CHK1, GADD45A, and MDM2 in OSCC tissue was significantly increased. The protein expression of the tumour suppressor gene P53 was also increased. Patients with high CHK1 and MDM2 expression levels had a reduced survival time and a poor prognosis, whereas patients with high GADD45A expression levels had a good prognosis. Our results indicate that high CHK1 expression is an independent risk factor for poor OSCC prognosis, and that CHK1 may be a potential target for OSCC clinical treatment. [ABSTRACT FROM AUTHOR]

Background: Aggressive metastasis of tumor cells assumed a constructive role in strengthening chemoresistance of tumors, so this investigation was intended to elucidate if lncRNA CCAT2 sponging downstream miR-424 regulated chemotolerance of glioma cells by boosting metastasis of glioma cells. Methods: One hundred and twenty-eight pairs of glioma tissues and corresponding adjacent tissues were resected from glioma patients during their operation, and we also purchased a series of glioma cell lines, including U251, U87, A172 and SHG44. Furthermore, pcDNA3.1-CCAT2, si-CCAT2, miR-424 mimic and miR-424 inhibitor were transfected into SHG44 and U251 cell lines, so as to evaluate impacts of CCAT2 and miR-424 on chemosensitivity of the glioma cells. Besides, proliferation, invasion and metastasis of the cells were determined through the implementation of colony formation assay, transwell assay and scratch assay. Results: Glioma tissues and cells were monitored with higher CCAT2 expression and lower miR-424 expression than adjacent normal tissues and NHA cell line (P< 0.05). Among the glioma cell lines, the SHG44 cell line showed the strongest resistance against teniposide, temozolomide and cisplatin (P< 0.05), whereas the U251 cell line was more sensitive to teniposide, temozolomide, vincristine and cisplatin than any other cell line (P< 0.05). Besides, pcDNA3.1-CCAT2 and miR-424 inhibitor could enhance tolerance of glioma cell lines against drugs (P< 0.05). Moreover, in-vitro transfection of si-CCAT2 and miR-424 mimic could significantly retard proliferation, invasion and migration of SHG44 and U251 cells (P< 0.05), and CCAT2 was found to negatively regulate miR-424 expression by sponging it (P< 0.05). In addition, CHK1 was deemed as the molecule targeted by upstream miR-424, and its overexpression can changeover the effects of miR-424 mimic on proliferation and metastasis of SHG44 and U251 cells. Conclusion: lncRNA CCAT2/miR-424/Chk1 axis might serve as a promising target for improving chemotherapeutic efficacies in glioma treatment. [ABSTRACT FROM AUTHOR]

Chromosomal instability not only has a negative effect on survival in triple-negative breast cancer, but also on the well treatable subgroup of luminal A tumors. This suggests a general mechanism independent of subtypes. Increased chromosomal instability (CIN) in triple-negative breast cancer (TNBC) is attributed to a defect in the DNA repair pathway homologous recombination. Homologous recombination (HR) prevents genomic instability by repair and protection of replication. It is unclear whether genetic alterations actually lead to a repair defect or whether superior signaling pathways are of greater importance. Previous studies focused exclusively on the repair function of HR. Here, we show that the regulation of HR by the intra-S-phase damage response at the replication is of overriding importance. A damage response activated by Ataxia telangiectasia and Rad3 related-checkpoint kinase 1 (ATR-CHK1) can prevent replication stress and leads to resistance formation. CHK1 thus has a preferred role over HR in preventing replication stress in TNBC. The signaling cascade ATR-CHK1 can compensate for a double-strand break repair error and lead to resistance of HR-deficient tumors. Established methods for the identification of HR-deficient tumors for Poly(ADP-Ribose)-Polymerase 1 (PARP1) inhibitor therapies should be extended to include analysis of candidates for intra-S phase damage response. [ABSTRACT FROM AUTHOR]

Checkpoint kinases 1 and 2 (CHK1 and CHK2) are important multifunctional proteins of the kinase family. Their main function is to regulate DNA replication and DNA damage response. If a cell is exposed to exogenous damage to its DNA, CHK1/CHK2 stops the cell cycle to give time to the cellular mechanisms to repair DNA breakage and apoptosis too, if the damage is not repairable to activate programmed cell death. CHK1/CHK2 plays a crucial role in the repair of recombination-mediated double-stranded DNA breaks. The other important functions performed by these proteins are the beginning of DNA replication, the stabilization of replication forks, the resolution of replication stress and the coordination of mitosis, even in the absence of exogenous DNA damage. Prexasertib (LY2606368) is a small ATP-competitive selective inhibitor of CHK1 and CHK2. In preclinical studies, prexasertib in monotherapy has shown to induce DNA damage and tumor cells apoptosis. The preclinical data and early clinical studies advocate the use of prexasertib in solid tumors both in monotherapy and in combination with other drugs (antimetabolites, PARP inhibitors and platinum-based chemotherapy). The safety and the efficacy of combination therapies with prexasertib need to be better evaluated in ongoing clinical trials. [ABSTRACT FROM AUTHOR]

DNA damage response (DDR) pathway prevents high level endogenous and environmental DNA damage being replicated and passed on to the next generation of cells via an orchestrated and integrated network of cell cycle checkpoint signalling and DNA repair pathways. Depending on the type of damage, and where in the cell cycle it occurs different pathways are involved, with the ATM-CHK2-p53 pathway controlling the G1 checkpoint or ATR-CHK1-Wee1 pathway controlling the S and G2/M checkpoints. Loss of G1 checkpoint control is common in cancer through TP53, ATM mutations, Rb loss or cyclin E overexpression, providing a stronger rationale for targeting the S/G2 checkpoints. This review will focus on the ATM-CHK2-p53-p21 pathway and the ATR-CHK1-WEE1 pathway and ongoing efforts to target these pathways for patient benefit. [ABSTRACT FROM AUTHOR]

While mammalian Chk1 kinase regulates replication origins, safeguards fork integrity and promotes fork progression, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A) in the kinase domain of fission yeast Chk1 abolishes the G1-M and S-M checkpoints with little impact on the G2-M arrest. This separation-of-function mutation strongly reduces the Rad3-dependent phosphorylation of Chk1 at serine 345 during logarithmic growth, but not when cells experience exogenous DNA damage. Loss of S173 lowers the restrictive temperature of a catalytic DNA polymerase epsilon mutant (cdc20.M10) and is epistatic with a mutation in DNA polymerase delta (cdc6.23) when DNA is alkylated by methyl-methanesulfate (MMS). The chk1-S173A allele is uniquely sensitive to high MMS concentrations where it displays a partial checkpoint defect. A complete checkpoint defect occurs only when DNA replication forks break in cells without the intra-S phase checkpoint kinase Cds1. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 (cdc10.V50) is impaired. We conclude that serine 173, which is equivalent to lysine 166 in the activation loop of human Chk1, is only critical in DNA polymerase mutants or when forks collapse in the absence of Cds1.

The effects of CHK1 inhibitor on cell cycle kinetics have not been fully investigated yet. In this study, we closely analyzed this kinetics using a CHK1 inhibitor (PF00477736) in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). This system allowed us to visualize cell cycle progression following CHK1 inhibitor treatment in real-time. FACS analysis showed that high levels of DNA damage as determined by γH2AX immunostaining was induced in S phase and that polyploid cells harboring the same levels of DNA damage appeared thereafter. Surprisingly, time-lapse imaging of Fucci fluorescence revealed that many cells entered M phase at once and exhibited prolonged mitosis; eventually progressing to G1 phase not accompanied by cytokinesis; this is an endomitosis-like event. Most of these cells then underwent S/G2 phases at least once, which corroborated the appearance of polyploid cells. However, a small fraction of cells with 2 N DNA content still remained 24 h after the treatment. When co-treated with MAD2 inhibitor, a core factor constituting spindle checkpoint, the 2 N DNA cell fraction disappeared and almost all cells exhibited endomitosis, leading to enhanced sensitivity. Detailed cell cycle analysis revealed that induction of an endomitosis-like event might be associated with CHK1 inhibitor-induced cell death in HeLa cells. • We studied CHK1 inhibitor-induced cell cycle kinetics in HeLa-Fucci cells. • DNA damage was induced in S phase and mitotic duration was elongated. • CHK1 inhibitor induced endomitosis and polyploidy. • MAD2 inhibitor co-treatment enhanced endomitosis and reduced cell survival. • Endomitosis may be linked to CHK1-induced cell death in HeLa cells. [ABSTRACT FROM AUTHOR]

In the presence of chromatin bridges, mammalian cells delay completion of cytokinesis (abscission) to prevent chromatin breakage or tetraploidization by regression of the cleavage furrow. This abscission delay is called "the abscission checkpoint" and is dependent on Aurora B kinase. Furthermore, cells stabilize the narrow cytoplasmic canal between the two daughter cells until the DNA bridges are resolved. Impaired abscission checkpoint signaling or unstable intercellular canals can lead to accumulation of DNA damage, aneuploidy, or generation of polyploid cells which are associated with tumourigenesis. However, the molecular mechanisms involved have only recently started to emerge. In this review, we focus on the molecular pathways of the abscission checkpoint and describe newly identified triggers, Aurora B-regulators and effector proteins in abscission checkpoint signaling. We also describe mechanisms that control intercellular bridge stabilization, DNA bridge resolution, or abscission checkpoint silencing upon satisfaction, and discuss how abscission checkpoint proteins can be targeted to potentially improve cancer therapy. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H 2 S), an endogenous gasotransmitter, exhibits the anxiolytic roles through its anti-inflammatory effects, although its underlying mechanisms remain largely elusive. Emerging evidence has documented that cell cycle checkpoint kinase 1 (Chk1)-regulated DNA damage plays an important role in the neurodegenerative diseases; however, there are few relevant reports on the research of Chk1 in neuropsychiatric diseases. Here, we aimed to investigate the regulatory role of H 2 S on Chk1 in lipopolysaccharide (LPS)-induced anxiety-like behavior focusing on inflammasome activation in the hippocampus. Cystathionine γ-lyase (CSE, a H 2 S-producing enzyme) knockout (CSE−/−) mice displayed anxiety-like behavior and activation of inflammasome-mediated inflammatory responses, manifesting by the increase levels of interleukin-1β (IL-1β), IL-6, and ionized calcium-binding adaptor molecule-1 (Iba-1, microglia marker) expression in the hippocampus. Importantly, expression of p-Chk1 and γ-H2AX (DNA damage marker) levels were also increased in the hippocampus of CSE−/− mice. LPS treatment decreased the expression of CSE and CBS while increased p-Chk1 and γ-H2AX levels and inflammasome-activated neuroinflammation in the hippocampus of mice. Moreover, p-Chk1 and γ-H2AX protein levels and cellular immunoactivity were significantly increased while CSE and CBS were markedly decreased in cultured BV2 cells followed by LPS treatment. Treatment of mice with GYY4137, a donor of H 2 S, inhibited LPS-induced increased in p-Chk1 and γ-H2AX levels, mitigated inflammasome activation and inflammatory responses as well as amelioration of anxiety-like behavior. Notably, SB-218078, a selective Chk1 inhibitor treatment attenuated the effect of LPS on inflammasome activation and inflammatory responses and the induction of anxiety-like behavior. Finally, STAT3 knockdown with AAV-STAT3 shRNA alleviated LPS-induced anxiety-like behavior and inhibited inflammasome activation in the hippocampus, and blockade of NLRP3 with MCC950 attenuated neuroinflammation induction and ameliorated LPS-induced anxiety-like behavior. Overall, this study indicates that downregulation of Chk1 activity by H 2 S activation may be considered as a valid strategy for preventing the progression of LPS-induced anxiety-like behavior. • CSE deletion increases Chk1 activation and induces anxiety-like behavior. • LPS suppresses CSE and CBS expression and promotes Chk1 activation. • H 2 S activation mitigates LPS-induced Chk1 activity and anxiety-like behavior. • Chk1 inhibition mitigates LPS-induced neuroinflammation and anxiety-like behavior. • Blockade of inflammasome attenuates LPS-induced anxiety-like behavior. [ABSTRACT FROM AUTHOR]

The human BK polyomavirus (BKPyV) is latent in the kidneys of most adults, but can be reactivated in immunosuppressed states, such as following renal transplantation. If left unchecked, BK polyomavirus nephropathy (PyVAN) and possible graft loss may result from viral destruction of tubular epithelial cells and interstitial fibrosis. When coupled with regular post-transplant screening, immunosuppression reduction has been effective in limiting BKPyV viremia and the development of PyVAN. Antiviral drugs that are safe and effective in combating BKPyV have not been identified but would be a benefit in complementing or replacing immunosuppression reduction. The present study explores inhibition of the host DNA damage response (DDR) as an antiviral strategy. Immunohistochemical and immunofluorescent analyses of PyVAN biopsies provide evidence for stimulation of a DDR in vivo. DDR pathways were also stimulated in vitro following BKPyV infection of low-passage human renal proximal tubule epithelial cells. The role of Chk1, a protein kinase known to be involved in the replication stress-induced DDR, was examined by inhibition with the small molecule LY2603618 and by siRNA-mediated knockdown. Inhibition of Chk1 resulted in decreased replication of BKPyV DNA and viral spread. Activation of mitotic pathways was associated with the reduction in BKPyV replication. Chk1 inhibitors that are found to be safe and effective in clinical trials for cancer should also be evaluated for antiviral activity against BKPyV.

Tight regulation of the eukaryotic cell cycle is paramount to ensure genomic integrity throughout life. Cell cycle checkpoints are present in each phase of the cell cycle and prevent cell cycle progression when genomic integrity is compromised. The G2 checkpoint is an intricate signaling network that regulates the progression of G2 to mitosis (M). We propose here a node‐based model of G2 checkpoint regulation, in which the action of the central CDK1–cyclin B1 node is determined by the concerted but opposing activities of the Wee1 and cell division control protein 25C (CDC25C) nodes. Phosphorylation of both Wee1 and CDC25C at specific sites determines their subcellular localization, driving them either toward activity within the nucleus or to the cytoplasm and subsequent ubiquitin‐mediated proteasomal degradation. In turn, this subcellular balance of the Wee1 and CDC25C nodes is directed by the action of the PLK1 and CHK1 nodes via what we have termed the ‘nuclear and cytoplasmic decision states’ of Wee1 and CDC25C. The proposed node‐based model provides an intelligible structure of the complex interactions that govern the decision to delay or continue G2/M progression. The model may also aid in predicting the effects of agents that target these G2 checkpoint nodes.

Cell division cycle 6 (CDC6), an androgen receptor (AR) target gene, is implicated in regulating DNA replication and checkpoint mechanisms. CDC6 expression is increased during prostate cancer (PCa) progression and positively correlates with AR in PCa tissues. AR or CDC6 knockdown, together with AZD7762, a Chk1/2 inhibitor, results in decreased TopBP1-ATR-Chk1 signaling and markedly increased ataxia-telangiectasia-mutated (ATM) phosphorylation, a biomarker of DNA damage, and synergistically increases treatment efficacy. Combination treatment with the AR signaling inhibitor enzalutamide (ENZ) and the Chk1/2 inhibitor AZD7762 demonstrates synergy with regard to inhibition of AR-CDC6-ATR-Chk1 signaling, ATM phosphorylation induction, and apoptosis in VCaP (mutant p53) and LNCaP-C4-2b (wild-type p53) cells. CDC6 overexpression significantly reduced ENZ- and AZD7762-induced apoptosis. Additive or synergistic therapeutic activities are demonstrated in AR-positive animal xenograft models. These findings have important clinical implications, since they introduce a therapeutic strategy for AR-positive, metastatic, castration-resistant PCa, regardless of p53 status, through targeting AR-CDC6-ATR-Chk1 signaling.

Inotuzumab ozogamicin (IO) is an anti-CD22 calicheamicin immunoconjugate that has been recently approved for the treatment of relapsed or refractory B-Acute Lymphoblastic Leukemia (r/r B-ALL). We employed both immortalized and primary cells derived from CD22-positive lymphoproliferative disorders to investigate the signaling pathways contributing to IO sensitivity or resistance. We found that the drug reduced the proliferation rate of CD22-positive cell lines expressing wild-type p53, but was remarkably less effective on cells exhibiting mutant p53. In addition, CD22-positive cells surviving IO were mostly blocked in the G2/M phase of the cell cycle because of Chk1 activation that, in the presence of a wild-type p53 background, led to p21 induction. When we combined IO with the Chk1 inhibitor UCN-01, we successfully abrogated IO-induced G2/M arrest regardless of the underlying p53 status, indicating that the DNA damage response triggered by IO is also modulated by p53-independent mechanisms. To establish a predictive value for p53 in determining IO responsiveness, we expressed mutant p53 in cell lines displaying the wild-type gene and observed an increase in IO IC50 values. Likewise, overexpression of an inducible wild-type p53 in cells natively presenting a mutant protein decreased their IC50 for IO. These results were also confirmed in primary CD22-positive cells derived from B-ALL patients at diagnosis and from patients with r/r B-ALL. Furthermore, co-treatment with IO and UCN-01 significantly increased cell death in primary cells expressing mutant p53. In summary, our findings suggest that p53 status may represent a biomarker predictive of IO efficacy in patients diagnosed with CD22-positive malignancies.

Despite the recent advancement in diagnosis and therapy, pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is still the most lethal cancer with a low five-year survival rate. There is an urgent need to develop new therapies to address this issue. In this study, we developed a treatment strategy by modifying tumor suppressor miRNAs, miR-15a and miR-194, with the chemotherapeutic gemcitabine (Gem) to create Gem-modified mimics, Gem-miR-15a and Gem-miR-194, respectively. In a panel of PDAC cell lines, we found that Gem-miR-15a and Gem-miR-194 induce cell-cycle arrest and apoptosis, and these mimics are potent inhibitors with IC 50 values up to several hundred fold less than their native counterparts or Gem alone. Furthermore, we found that Gem-miR-15a and Gem-miR-194 retained miRNA function by downregulating the expression of several key targets including WEE1, CHK1, BMI1, and YAP1 for Gem-miR-15a, and FOXA1 for Gem-miR-194. We also found that our Gem-modified miRNA mimics exhibit an enhanced efficacy compared to Gem in patient-derived PDAC organoids. Furthermore, we observed that Gem-miR-15a significantly inhibits PDAC tumor growth in vivo without observing any noticeable signs of toxicity. Overall, our results demonstrate the therapeutic potential of Gem-modified miRNAs as a treatment strategy for PDAC., Competing Interests: J.J. and A.F. have filed a patent for Gem-modified miRNA mimics. J.J. is a scientific co-founder of Curamir Therapeutics. The remaining authors declare no competing interests., (© 2024 The Author(s).)

The ATR/Chk1 pathway is an important regulator of cell cycle progression, notably upon genotoxic stress where it can detect a large variety of DNA alterations and induce a transient cell cycle arrest that promotes DNA repair. In addition to its role in DNA damage response (DDR), Chk1 is also active during a non-perturbed S phase and contributes to prevent a premature entry into mitosis with an incompletely replicated genome, meaning the ATR/Chk1 pathway is an integral part of the cell cycle machinery that preserves genome integrity during cell growth. We recently developed a FRET-based Chk1 kinase activity reporter to directly monitor and quantify the kinetics of Chk1 activation in live single cell imaging assays with unprecedented sensitivity and time resolution. This tool allowed us to monitor Chk1 activity dynamics over time during a normal S phase and following genotoxic stress, and to elucidate the underlying mechanisms leading to its activation. Here, we review available fluorescent tools to study the interplay of cell cycle progression, DNA damage and DDR in individual live cells, and present the full protocol and image analysis pipeline to monitor Chk1 activity in two imaging assays., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Checkpoint kinases Chk1, Chk2, Wee1 are playing a key role in DNA damage response and genomic integrity. Cancer-associated mutations identified in human Chk1, Chk2, and Wee1 were retrieved to understand the function associated with the mutation and also alterations in the folding pattern. Therefore, an attempt has been made to identify deleterious effect of variants using in silico and structure-based approach. Variants of uncertain significance for Chk1, Chk2, and Wee1 were retrieved from different databases and four prediction servers were employed to predict pathogenicity of mutations. Further, Interpro, I-Mutant 3.0, Consurf, TM-align, and have (y)our protein explained were used for comprehensive study of the deleterious effects of variants. The sequences of Chk1, Chk2, and Wee1 were analyzed using Clustal Omega, and the three-dimensional structures of the proteins were aligned using TM-align. The molecular dynamics simulations were performed to explore the differences in folding pattern between Chk1, Chk2, Wee1 wild-type, and mutant protein and also to evaluate the structural integrity. Thirty-six variants in Chk1, 250 Variants in Chk2, and 29 in Wee1 were categorized as pathogenic using in silico prediction tools. Furthermore, 25 mutations in Chk1, 189 in Chk2, and 14 in Wee1 were highly conserved, possessing deleterious effect and also influencing the protein structure and function. These identified mutations may provide underlying genetic intricacies to serve as potential targets for therapeutic inventions and clinical management., (© 2023 Wiley Periodicals LLC.)

In response to perturbed DNA replication, ATR (ataxia telangiectasia and Rad3-related) kinase is activated to initiate the checkpoint signaling necessary for maintaining genome integrity and cell survival. To better understand the signaling mechanism, we carried out a large-scale genetic screen in fission yeast looking for mutants with enhanced sensitivity to hydroxyurea. From a collection of 370 primary mutants, we found a few mutants in which Rad3 (ATR ortholog)-mediated phospho-signaling was significantly compromised. One such mutant carried an uncharacterized mutation in tel2, a gene encoding an essential and highly conserved eukaryotic protein. Previous studies in various biological models have shown that Tel2 mainly functions in Tel2-Tti1-Tti2 (TTT) complex that regulates the steady-state levels of all phosphatidylinositol 3-kinase-like protein kinases, including ATR. We show here that although the levels of Rad3 and Rad3-mediated phospho-signaling in DNA damage checkpoint were moderately reduced in the tel2 mutant, the phospho-signaling in the DNA replication checkpoint was almost completely eliminated. In addition, the tel2 mutation caused telomere shortening. Since the interactions of Tel2 with Tti1 and Tti2 were significantly weakened by the mutation, destabilization of the TTT complex likely contributes to the observed checkpoint and telomere defects. [ABSTRACT FROM AUTHOR]

Objective To investigate the role of synaptic binding protein 7 (Syt-7) in hepatocellular carcinoma ( HCC) by detecting its expression and elucidate its potential mechanism. Methods The expression levels of Syt-7 mRNA in liver cancer cells and normal liver cells were detected by RT-qPCR. Celigo cell count, MTT detection, clone detection and cell cycle analysis were used to evaluate the effect of low Syt-7 in vitro. Tumor formation in vivo was evaluated by nude mouse model. Application PathScan Antibody Array Kit to evaluate potential molecular mechanisms. Results The mRNA of Syt-7 was highly expressed in hepatocellular carcinoma cells Huh-7 and Hep3B, moderately expressed in hepatocellular carcinoma cells SMMC-7721, HepG2 and BEL-7402, and poorly expressed in normal hepatocellular carcinoma cells L-O2. Functional experiments showed that knockdown of Syt-7 significantly inhibited cell proliferation and induced cell cycle arrest by increasing phosphorylation of Chkl and P53. In addition, knock down of Syt-7 significantly inhibited tumor formation in nude mice. Conclusion Syt-7 plays an important role in tumor formation and progression of HCC and may be a new therapeutic target for HCC. [ABSTRACT FROM AUTHOR]

Cyclins are central engines of cell cycle progression in conjunction with cyclin‐dependent kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner with a CDK, but instead forms via its F‐box domain an SCF (Skp1‐Cul1‐F‐box)‐type E3 ubiquitin ligase module. Although various substrates of cyclin F have been identified, the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging them with more than 180 different kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. Replication catastrophe depends on accumulation of the transcription factor E2F1 in cyclin F‐depleted cells. We find that SCF‐cyclin F controls E2F1 ubiquitylation and degradation during the G2/M phase of the cell cycle and upon challenging cells with Chk1 inhibitors. Thus, Cyclin F restricts E2F1 activity during the cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors. Synopsis: A kinase inhibitor screen for synthetic lethality in cells lacking the ubiquitin ligase adaptor Cyclin F leads to the identification of E2F1 as new SCF‐Cyclin F substrate, whose unrestricted activity promotes catastrophic replication stress upon checkpoint suppression with Chk1 inhibitors. A screen to identify vulnerabilities of cells lacking cyclin F identifies Chk1 inhibitors as primary hits.Cyclin F loss predisposes cells to the toxic effect of Chk1i by inducing DNA replication catastrophe.Aberrant accumulation of E2F1 upon cyclin F loss promotes DNA damage upon Chk1 inhibition.Lack of cyclin F prevents degradation of E2F1 in G2/M and upon Chk1 inhibitors.A non‐degradable E2F1 lacking a CY motif induces cell death and DNA replication stress upon Chk1 inhibition. [ABSTRACT FROM AUTHOR]

Human papillomavirus 8 (HPV8) is associated with the development of squamous cell carcinoma (SCC) of the skin. HPV‐infected keratinocytes are able to override normal checkpoint control mechanisms and sustain cell cycle activity, allowing for synthesis of cellular proteins necessary for viral genome amplification. To study how HPV8 may disrupt cell cycle control, we analyzed the impact of HPV8 early gene expression on one of the key regulators of cell cycle and DNA damage response, checkpoint kinase‐1 (CHK1). We found that expression of E1, E1̂E4, E2, E6 or E7 individually did not affect CHK1; however, keratinocytes expressing the complete early genome region (CER) of HPV8 showed a profound loss of CHK1 protein levels, that proved to be mediated by E6E7 co‐expression. Neither CHK1 promoter regulation nor the ubiquitin‐proteasome pathway are involved in HPV8‐mediated CHK1 repression. However, CHK1 protein repression in organotypic skin cultures was paralleled by downregulation of the autophagy marker LC3B. Treatment of HPV8‐CER expressing cells with the autophagy inhibitor Bafilomycin A1 rescued CHK1 expression and led to LC3B accumulation. Taken together, our data implicate that CHK1 autophagic degradation is enhanced by HPV8, which may contribute to the oncogenic potential of the virus. What's new? While human papillomavirus type 8 (HPV8) is suspected of playing a role in cutaneous squamous cell carcinoma, the contributions of HPV8 to skin tumor development remain unknown. Here, expression of the complete early genome region of HPV8 was associated with a significant reduction in checkpoint kinase‐1 (CHK1) protein levels. CHK1 downregulation by HPV8 was dependent on E6E7 oncoprotein co‐expression and autophagic processes. The data demonstrate a novel oncogenic effect of HPV8 in keratinocytes, whereby CHK1 absence enables virus‐infected cells to survive in a replicative state, predisposing them to the accumulation of DNA damage from ultraviolet radiation. [ABSTRACT FROM AUTHOR]

Fbxo6 (also called FBG2) is a critical component of the evolutionarily conserved ubiquitin–protein ligase complex SCF (Skp1/Cdc53‐Cullin1/F‐box). Previous studies have demonstrated that Fbxo6 facilitates the growth and proliferation but inhibits the apoptosis and invasion of gastric cancer cells. However, the role of Fbxo6 in non‐small cell lung cancer (NSCLC) is still not clear. Our results revealed that Fbxo6 expression is correlated with early TNM stage and favorable overall survival of NSCLC patients. Further in vitro experiments showed that Fbxo6 inhibits proliferation, facilitates apoptosis and promotes the sensitivity of cisplatin via decreased expression and phosphorylation of Chk1. Thus, Fbxo6 may be a useful prognosis marker and therapeutic target to overcome the chemoresistance of cisplatin‐based chemotherapy agents in NSCLC patients. [ABSTRACT FROM AUTHOR]

Purpose: Triple-negative breast cancers (TNBC) are often resistant to treatment with ionizing radiation (IR). We sought to investigate whether pharmacologic inhibition of Chk1 kinase, which is commonly overexpressed in TNBC, preferentially sensitizes TNBC cells to IR. Methods: Ten breast cancer cell lines were screened with small molecule inhibitors against Chk1 and other kinases. Chk1 inhibition was also tested in isogenic KRAS mutant or wild-type cancer cells. Cellular radiosensitization was measured by short-term and clonogenic survival assays and by staining for the DNA double-strand break (DSB) marker γ-H2AX. Radiosensitization was also assessed in breast cancer biopsies using an ex vivo assay. Aurora B kinase-dependent mitosis-like chromatin condensation, a marker of radioresistance, was detected using a specific antibody against co-localized phosphorylation of serine 10 and trimethylation of lysine 9 on histone 3 (H3K9me3/S10p). Expression of CHEK1 and associated genes was evaluated in TNBC and lung adenocarcinoma. Results: Inhibition of Chk1 kinase preferentially radiosensitized TNBC cells in vitro and in patient biopsies. Interestingly, TNBC cells displayed lower numbers of IR-induced DSBs than non-TNBC cells, correlating with their observed radioresistance. We found that Chk1 suppressed IR-induced DSBs in these cells, which was dependent on H3K9me3/S10p—a chromatin mark previously found to indicate radioresistance in KRAS mutant cancers. Accordingly, the effects of Chk1 inhibition in TNBC were reproduced in KRAS mutant but not wild-type cells. We also observed co-expression of genes in this Chk1 chromatin pathway in TNBC and KRAS mutant lung cancers. Conclusions: Chk1 promotes an unexpected, common phenotype of chromatin-dependent DSB suppression in radioresistant TNBC and KRAS mutant cancer cells, providing a direction for future investigations into overcoming the treatment resistance of TNBC. [ABSTRACT FROM AUTHOR]

Inotuzumab ozogamicin (IO) is an anti-CD22 calicheamicin immunoconjugate that has been recently approved for the treatment of relapsed or refractory B-Acute Lymphoblastic Leukemia (r/r B-ALL). We employed both immortalized and primary cells derived from CD22-positive lymphoproliferative disorders to investigate the signaling pathways contributing to IO sensitivity or resistance. We found that the drug reduced the proliferation rate of CD22-positive cell lines expressing wild-type p53, but was remarkably less effective on cells exhibiting mutant p53. In addition, CD22-positive cells surviving IO were mostly blocked in the G2/M phase of the cell cycle because of Chk1 activation that, in the presence of a wild-type p53 background, led to p21 induction. When we combined IO with the Chk1 inhibitor UCN-01, we successfully abrogated IO-induced G2/M arrest regardless of the underlying p53 status, indicating that the DNA damage response triggered by IO is also modulated by p53-independent mechanisms. To establish a predictive value for p53 in determining IO responsiveness, we expressed mutant p53 in cell lines displaying the wild-type gene and observed an increase in IO IC50 values. Likewise, overexpression of an inducible wild-type p53 in cells natively presenting a mutant protein decreased their IC50 for IO. These results were also confirmed in primary CD22-positive cells derived from B-ALL patients at diagnosis and from patients with r/r B-ALL. Furthermore, co-treatment with IO and UCN-01 significantly increased cell death in primary cells expressing mutant p53. In summary, our findings suggest that p53 status may represent a biomarker predictive of IO efficacy in patients diagnosed with CD22-positive malignancies. [ABSTRACT FROM AUTHOR]

Claspin was discovered as a Chk1‐interacting protein necessary for Chk1 phosphorylation and activation by the upstream kinase, ATR, in response to DNA synthesis inhibition in Xenopus oocyte extracts. Subsequent investigations have defined a molecular model in which Claspin acts as an adaptor or scaffold protein to facilitate activation of Chk1 by ATR within a multiprotein complex that forms on single‐stranded DNA at stalled replication forks and sites of DNA damage. Interestingly, Claspin is an unstable protein whose degradation via the proteasome is tightly regulated via ubiquitination and controlled by multiple ubiquitin ligases and deubiquitinases. As a result, Claspin levels fluctuate during the cell cycle, contributing to the regulation of checkpoint proficiency and playing a key role in terminating checkpoint‐mediated cell cycle arrest. In addition to its role in signalling genotoxic stress, Claspin is required to maintain normal rates of replication fork progression during unperturbed DNA replication and may contribute to the regulation of replication origin firing. Consistent with this, Claspin can bind directly to DNA, with particular affinity for branched or forked molecules, and it interacts with multiple protein components of the replisome. As expected for a protein with key roles in checkpoint signalling and genome duplication, aberrations of Claspin expression and structure have been observed in cancer. Claspin is furthermore targeted to facilitate viral replication and plays a role in suppressing cellular DNA synthesis in response to nongenotoxic endoplasmic reticulum stress. Here, we review the functions and regulation of Claspin with a focus on areas of active research. Claspin is an adaptor molecule that facilitates activation of the checkpoint effector kinase, Chk1, by its upstream activator ATR within a multiprotein complex that forms at sites of DNA damage or stalled replication. Claspin is also thought to play an important role in normal replication, and may be a target for certain nongenotoxic stress signalling pathways that inhibit DNA synthesis. [ABSTRACT FROM AUTHOR]

The ATR/CHK1 pathway is a key effector of cellular response to DNA damage and therefore is a critical regulator of genomic stability. While the ATR/CHK1 pathway is often inactivated by mutations, CHK1 itself is rarely mutated in human cancers. Thus, cellular levels of CHK1 likely play a key role in the maintenance of genomic stability and preventing tumorigenesis. Glucose deprivation is observed in many solid tumors due to high glycolytic rates of cancer cells and insufficient vascularization, yet cancer cells have devised mechanisms to survive in conditions of low glucose. Although CHK1 degradation through the ubiquitin–proteasome pathway following glucose deprivation has been previously reported, the detailed molecular mechanisms remain elusive. Here, we show that CHK1 is ubiquitinated and degraded upon glucose deprivation by the Skp1‐Cullin‐F‐box (β‐TrCP) E3 ubiquitin ligase. Specifically, CHK1 contains a β‐TrCP recognizable degron domain, which is phosphorylated by AMPK in response to glucose deprivation, allowing for β‐TrCP to recognize CHK1 for subsequent ubiquitination and degradation. Our results provide a novel mechanism by which glucose metabolism regulates a DNA damage effector, and imply that glucose deprivation, which is often found in solid tumor microenvironments, may enhance mutagenesis, clonal expansion, and tumor progression by triggering CHK1 degradation. Glucose deprivation is observed in many solid tumors, and cancer cells have devised mechanisms to survive in conditions of low glucose. Here, we show that CHK1, a critical regulator of genomic stability, is ubiquitinated and degraded upon glucose deprivation by the β‐TrCP E3 ubiquitin ligase. Our results indicate that glucose deprivation may enhance tumorigenesis through inactivation of the CHK1 pathway. [ABSTRACT FROM AUTHOR]

OBJECTIVE: This study aims to investigate effects of checkpoint kinase, mediator of DNA damage checkpoint 1 (MDC1) and p53-binding protein 1 (53BP1) silencing on p53, checkpoint kinase 1 and 2 (CHK1 and CHK2), and CHK2-T68 expression. MATERIALS AND METHODS: Eca109 cells were divided into untransfected Eca109, Blank-vector, MDC1-RNAi transfection, and 53BP1-RNAi transfection group. Streptavidin- peroxidase (SP) immunohistochemical assay was used to examine CHK2-T68 expression. About 4 groups were used to establish esophageal carcinoma nude-mouse models, and assigned as Eca-109 control (or Eca-109 plus 15 Gy γ-rays irradiation, Eca-109+IR), Blank-vector (or Blank-vecor+IR), 53BP1-RNAi (or 53BP1- RNAi+IR), and MDC1-RNAi group (or MDC1- RNAi+IR group) by injecting. The expression of p53, CHK1, CHK2 were evaluated using SP immunohistochemical assay. RESULTS: 53BP1 and MDC1 down-regulation significantly inhibited expression of CHK2-T68 in Eca-109 cells compared to untreated group (p<0.05). There were significant differences for CHK2-T68 expressions in different time and groups (p<0.05). 53BP1 down-regulation significantly reduced p53 and enhanced CHK1 and CHK2 expression compared to that of Eca-109 control group (p<0.05) in Eca-109 cells. 53BP1 down-regulation significantly regulated CHK1, CHK2, and p53 in xenograft nude mice models exposed to γ-ray irradiation compared to that of untreated group (p<0.05). p53 was negatively correlated with CHK1 and CHK2 in xenograft nude mice models. CONCLUSIONS: 53BP1 regulated the cell cycle arrest by modulating p53, CHK1, and CHK2 expression in both Eca-109 cells and xenograft nude mice models. [ABSTRACT FROM AUTHOR]

Highlights • ATR kinase regulates intracellular hydrogen sulfide concentrations pharmacologic inhibition ATR lowers cellular hydrogen sulfide levels and hydrogen sulfide levels are also lower syngeneic cells carrying a knock-in hypomorphic ATR Seckel mutant ATR protein. • ATR serine 435 phosphorylation is suppressed by exogenous hydrogen and increased by a hydrogen sulfide synthesis inhibitor, indicating that ATR may be regulated by hydrogen sulfide concentrations. • ATR kinase activity is modulated by hydrogen sulfide, as treatment of ATR wild type cells with a hydrogen sulfide synthesis inhibitor potentiates CHK1 protein phosphorylation by the ATR kinase. • Lastly, we present data indicating that hydrogen sulfide may influence genomic stability in conjunction with ATR kinase activity. • We believe these are novel findings and show a new function for the ATR kinase. Abstract The ataxia telangiectasia-mutated and Rad3-related (ATR) serine/threonine kinase plays a central role in the repair of replication-associated DNA damage, the maintenance of S and G2/M-phase genomic stability, and the promotion of faithful mitotic chromosomal segregation. A number of stimuli activate ATR, including persistent single-stranded DNA at stalled replication folks, R loop formation, hypoxia, ultraviolet light, and oxidative stress, leading to ATR-mediated protein phosphorylation. Recently, hydrogen sulfide (H 2 S), an endogenous gasotransmitter, has been found to regulate multiple cellular processes through complex redox reactions under similar cell stress environments. Three enzymes synthesize H 2 S: cystathionine-β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase. Since H 2 S can under some conditions cause DNA damage, we hypothesized that ATR activity may regulate cellular H 2 S concentrations and H 2 S-syntheszing enzymes. Here we show that human colorectal cancer cells carrying biallelic knock-in hypomorphic ATR mutations have lower cellular H 2 S concentrations than do syngeneic ATR wild-type cells, and all three H 2 S-synthesizing enzymes show lower protein expression in the ATR hypomorphic mutant cells. Additionally, ATR serine 428 phosphorylation is altered by H 2 S donor and H 2 S synthesis enzyme inhibition, while the oxidative-stress induced phosphorylation of the ATR-regulated protein CHK1 on serine 345 is increased by H 2 S synthesis enzyme inhibition. Lastly, inhibition of H 2 S production potentiated oxidative stress-induced double-stranded DNA breaks in the ATR hypomorphic mutant compared to ATR wild-type cells. Our findings demonstrate that the ATR kinase regulates and is regulated by H 2 S. [ABSTRACT FROM AUTHOR]

Chemotherapy resistance represents a major issue associated with gastric cancer (GC) treatment, and arises through multiple mechanisms, including modulation of the cell-cycle check point. Several ubiquitin kinases, including RING finger protein 138 (RNF138), have been reported to mediate the G2/M phase arrest. In this study, we investigated the role of RNF138 in the development of cisplatin resistance of two GC cell lines. We show that RNF138 levels are higher in cisplatin-resistant cell lines, compared with cisplatin-sensitive cells, and RNF138 expression was elevated during drug withdrawal following the cisplatin treatment. Using gene overexpression and silencing, we analyzed the impact of altering RNF138 level on GC cell viability, apoptosis, and cell cycle phenotypes in two isogenic cisplatin-sensitive and resistant cell lines. We show that RNF138 overexpression increased GC cell viability, decreased apoptosis and delayed cell cycle progression in the cisplatin-sensitive GC cells. Conversely, RNF138 silencing produced opposite phenotypes in the cisplatin-resistant cells. Moreover, RNF138-dependent phosphorylation of Chk1 was seen in GC cells, indicating a novel connection between cisplatin-induced DNA damage and apoptosis. Collectively, these data suggest that RNF138 modulates the cisplatin resistance in the GC cells, thus serving as a potential drug target to challenge chemotherapy failure. In addition, RNF138 can also be used as a marker to monitor the development of cisplatin resistance in GC treatment. [ABSTRACT FROM AUTHOR]

RNA-binding proteins are frequently dysregulated in human cancer and able to modulate tumor cell proliferation as well as tumor metastasis through post-transcriptional regulation on target genes. Abnormal DNA damage response and repair mechanism are closely related to genome instability and cell transformation. Here, we explore the function of the RNA-binding protein muscleblind-like splicing regulator 2 (MBNL2) on tumor cell proliferation and DNA damage response. Transcriptome and gene expression analysis show that the PI3K/AKT pathway is enriched in MBNL2-depleted cells, and the expression of cyclin-dependent kinase inhibitor 1A (p21CDKN1A) is significantly affected after MBNL2 depletion. MBNL2 modulates the mRNA and protein levels of p21, which is independent of its canonical transcription factor p53. Moreover, depletion of MBNL2 increases the phosphorylation levels of checkpoint kinase 1 (Chk1) serine 345 (S345) and DNA damage response, and the effect of MBNL2 on DNA damage response is p21-dependent. MBNL2 would further alter tumor cell fate after DNA damage, MBNL2 knockdown inhibiting DNA damage repair and DNA damage-induced senescence, but promoting DNA damage-induced apoptosis.

Patients diagnosed with basal-like breast cancer suffer from poor prognosis and limited treatment options. There is an urgent need to identify new targets that can benefit patients with basal-like and claudin-low (BL-CL) breast cancers. We screened fractions from our Marine Invertebrate Compound Library (MICL) to identify compounds that specifically target BL-CL breast cancers. We identified a previously unreported trisulfated sterol, i.e., topsentinol L trisulfate (TLT), which exhibited increased efficacy against BL-CL breast cancers relative to luminal/HER2+ breast cancer. Biochemical investigation of the effects of TLT on BL-CL cell lines revealed its ability to inhibit activation of AMP-activated protein kinase (AMPK) and checkpoint kinase 1 (CHK1) and to promote activation of p38. The importance of targeting AMPK and CHK1 in BL-CL cell lines was validated by treating a panel of breast cancer cell lines with known small molecule inhibitors of AMPK (dorsomorphin) and CHK1 (Ly2603618) and recording the increased effectiveness against BL-CL breast cancers as compared with luminal/HER2+ breast cancer. Finally, we generated a drug response gene-expression signature and projected it against a human tumor panel of 12 different cancer types to identify other cancer types sensitive to the compound. The TLT sensitivity gene-expression signature identified breast and bladder cancer as the most sensitive to TLT, while glioblastoma multiforme was the least sensitive.

Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds. [Display omitted] • Genomic screen identifies mitotic regulation as a key decitabine resistance pathway • Clinical doses of decitabine induce extensive mitotic disruption in myeloid tumors • Decitabine-induced covalent binding of DNMT1 to DNA causes mitotic disruption • Inhibition of ATR-CHK1 synergizes with decitabine by impairing mitotic fidelity Yabushita et al. perform a genome-wide CRISPR activation screen to identify core regulators of resistance to decitabine in myeloid tumors. This screen reveals the potent activity of clinical-dose decitabine to disrupt mitosis via DNMT1-DNA adducts, challenging the prevailing notion that DNA demethylation is the primary mechanism of action of decitabine. [ABSTRACT FROM AUTHOR]