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1.
Oncol Res ; 32(6): 1021-1030, 2024.
Article in English | MEDLINE | ID: mdl-38827321

ABSTRACT

Background: Apolipoprotein B mRNA editing catalytic polypeptide (APOBEC), an endogenous mutator, induces DNA damage and activates the ataxia telangiectasia and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) pathway. Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer (MIBC), it has a poor survival rate. Therefore, this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B (APOBEC3B) expressing MIBC. Methods: Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC. The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis. Western blot analysis was performed to confirm differences in phosphorylated Chk1 (pChk1) expression according to the APOBEC3B expression. Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin. Conclusion: There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC. Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels. Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression. Compared to cisplatin single treatment, combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression. Conclusion: Our study shows that APOBEC3B's higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition. This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.


Subject(s)
Ataxia Telangiectasia Mutated Proteins , Cisplatin , Cytidine Deaminase , Minor Histocompatibility Antigens , Urinary Bladder Neoplasms , Humans , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Cisplatin/pharmacology , Cisplatin/therapeutic use , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Cell Line, Tumor , Male , Minor Histocompatibility Antigens/metabolism , Minor Histocompatibility Antigens/genetics , Middle Aged , Female , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/antagonists & inhibitors , Checkpoint Kinase 1/genetics , Apoptosis , Aged , Neoplasm Invasiveness , Cell Proliferation , Cell Survival/drug effects
2.
Sci Rep ; 14(1): 11788, 2024 05 23.
Article in English | MEDLINE | ID: mdl-38783016

ABSTRACT

Fascaplysin is a red cytotoxic pigment with anticancer properties isolated from the marine sponge Fascaplysinopsis sp. Recently, structure-activity relationship analysis reported by our group suggested that selective cytotoxicity of fascaplysin derivatives towards tumor cells negatively correlates with their ability to intercalate into DNA. To validate this hypothesis, we synthesized 6- and 7-tert-butylfascaplysins which reveal mitigated DNA-intercalating properties. These derivatives were found to be strongly cytotoxic to drug-resistant human prostate cancer cells, albeit did not demonstrate improved selectivity towards cancer cells when compared to fascaplysin. At the same time, kinome analysis suggested an activation of CHK1/ATR axis in cancer cells shortly after the drug exposure. Further experiments revealed induction of replication stress that is eventually converted to the toxic DNA double-strand breaks, resulting in caspase-independent apoptosis-like cell death. Our observations highlight new DNA-targeting effect of some fascaplysin derivatives and indicate more complex structure-activity relationships within the fascaplysin family, suggesting that cytotoxicity and selectivity of these alkaloids are influenced by multiple factors. Furthermore, combination with clinically-approved inhibitors of ATR/CHK1 as well as testing in tumors particularly sensitive to the DNA damage should be considered in further studies.


Subject(s)
Antineoplastic Agents , Checkpoint Kinase 1 , Humans , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/antagonists & inhibitors , Indoles/pharmacology , Indoles/chemistry , Apoptosis/drug effects , Structure-Activity Relationship , Male , Ataxia Telangiectasia Mutated Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , DNA/metabolism , Animals , DNA Breaks, Double-Stranded/drug effects , Quaternary Ammonium Compounds , Carbolines , Indolizines
3.
Cells ; 13(10)2024 May 17.
Article in English | MEDLINE | ID: mdl-38786089

ABSTRACT

Resistance to olaparib is the major obstacle in targeted therapy for ovarian cancer (OC) with poly(ADP-ribose) polymerase inhibitors (PARPis), prompting studies on novel combination therapies to enhance olaparib efficacy. Despite identifying various mechanisms, understanding how OC cells acquire PARPi resistance remains incomplete. This study investigated microRNA (miRNA) expression in olaparib-sensitive (PEO1, PEO4) and previously established olaparib-resistant OC cell lines (PEO1-OR) using high-throughput RT-qPCR and bioinformatic analyses. The role of miRNAs was explored regarding acquired resistance and resensitization with the ATR/CHK1 pathway inhibitors. Differentially expressed miRNAs were used to construct miRNA-mRNA regulatory networks and perform functional enrichment analyses for target genes with miRNet 2.0. TCGA-OV dataset was analyzed to explore the prognostic value of selected miRNAs and target genes in clinical samples. We identified potential processes associated with olaparib resistance, including cell proliferation, migration, cell cycle, and growth factor signaling. Resensitized PEO1-OR cells were enriched in growth factor signaling via PDGF, EGFR, FGFR1, VEGFR2, and TGFßR, regulation of the cell cycle via the G2/M checkpoint, and caspase-mediated apoptosis. Antibody microarray analysis confirmed dysregulated growth factor expression. The addition of the ATR/CHK1 pathway inhibitors to olaparib downregulated FGF4, FGF6, NT-4, PLGF, and TGFß1 exclusively in PEO1-OR cells. Survival and differential expression analyses for serous OC patients revealed prognostic miRNAs likely associated with olaparib resistance (miR-99b-5p, miR-424-3p, and miR-505-5p) and resensitization to olaparib (miR-324-5p and miR-424-3p). Essential miRNA-mRNA interactions were reconstructed based on prognostic miRNAs and target genes. In conclusion, our data highlight distinct miRNA profiles in olaparib-sensitive and olaparib-resistant cells, offering molecular insights into overcoming resistance with the ATR/CHK1 inhibitors in OC. Moreover, some miRNAs might serve as potential predictive signature molecules of resistance and therapeutic response.


Subject(s)
Ataxia Telangiectasia Mutated Proteins , BRCA2 Protein , Checkpoint Kinase 1 , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , MicroRNAs , Ovarian Neoplasms , Phthalazines , Piperazines , RNA, Messenger , Humans , Phthalazines/pharmacology , Phthalazines/therapeutic use , MicroRNAs/genetics , MicroRNAs/metabolism , Female , Piperazines/pharmacology , Piperazines/therapeutic use , Ovarian Neoplasms/genetics , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/genetics , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/drug effects , Cell Line, Tumor , Gene Regulatory Networks/drug effects , Ataxia Telangiectasia Mutated Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Signal Transduction/drug effects
4.
Aging (Albany NY) ; 16(10): 9168-9187, 2024 May 30.
Article in English | MEDLINE | ID: mdl-38819231

ABSTRACT

Stress is an important initiating factor in promoting Alzheimer's disease (AD) pathogenesis. However, the mechanism by which stress induces AD-like cognitive impairment remains to be clarified. Here, we demonstrate that DNA damage is increased in stress hormone Corticotropin-releasing factor (CRF)-treated cells and in brains of mice exposed to chronic restraint stress. Accumulation of DNA damage drives activation of cell cycle checkpoint protein kinase 1 (Chk1), upregulation of cancerous inhibitor of PP2A (CIP2A), tau hyperphosphorylation, and Aß overproduction, eventually resulting in synaptic impairment and cognitive deficits. Pharmacological intervention targeting Chk1 by specific inhibitor and DNA damage by vitamin C, suppress DNA damage-Chk1-CIP2A signaling pathway in chronic stress animal model, which in turn attenuate AD-like pathologies, synaptic impairments and cognitive deficits. Our study uncovers a novel molecular mechanism of stress-induced AD-like pathologies and provides effective preventive and therapeutic strategies targeting this signaling pathway.


Subject(s)
Alzheimer Disease , Checkpoint Kinase 1 , DNA Damage , Signal Transduction , Stress, Psychological , Animals , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Checkpoint Kinase 1/metabolism , Mice , Stress, Psychological/complications , Stress, Psychological/metabolism , Male , Humans , Disease Models, Animal , Membrane Proteins/metabolism , Membrane Proteins/genetics
5.
Nat Commun ; 15(1): 4667, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38821952

ABSTRACT

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.


Subject(s)
Auranofin , Carcinoma, Non-Small-Cell Lung , Checkpoint Kinase 1 , Lung Neoplasms , Oxidation-Reduction , Thioredoxins , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/antagonists & inhibitors , Humans , Oxidation-Reduction/drug effects , Thioredoxins/metabolism , Cell Line, Tumor , Auranofin/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Protein Kinase Inhibitors/pharmacology , Ribonucleoside Diphosphate Reductase/metabolism , Ribonucleoside Diphosphate Reductase/genetics , Ribonucleotide Reductases/metabolism , Ribonucleotide Reductases/antagonists & inhibitors , Drug Synergism , Animals
6.
PLoS One ; 19(4): e0302075, 2024.
Article in English | MEDLINE | ID: mdl-38669256

ABSTRACT

Endometrial cancer is the most prevalent gynecologic malignancy with a high risk of recurrence. Local recurrence occurs in 7-20% of patients with treated stage I cancer within 3 years after primary treatment. In this study, we found significantly elevated mRNA expression levels of the oncoprotein KRAS, along with two replicative stress markers, ATR and CHEK1, in samples of endometrial carcinomas of endometrium (ECE) from patients with relapse. In contrast, mRNA expression levels of the studied genes were low and uniform in samples from patients without relapse. Elevated levels of KRAS protein and the phosphorylated form of ATR/CHEK1 were distinguishing features of recurrent ECE. A strong positive correlation was found between elevated mRNA and protein levels of the studied molecules. Elevated KRAS protein levels are characteristic of poorly differentiated (G3) endometrial carcinomas with deep myometrial invasion in patients without recurrence. In contrast, in patients with recurrence, higher protein levels of KRAS, pATR and pCHEK1 were observed in samples of G1-2 endometrial carcinomas, with statistically significant differences confirmed for pATR. High pCHEK1 protein levels are associated with deep tumor invasion in the myometrium among patients with recurrence. ROC analysis confirmed that evaluating the specificity and sensitivity of KRAS, pATR and pCHEK1 predicts recurrence development in patients with ECE. Our findings indicate that markers of replicative stress may play a significant role in ECE pathogenesis. Determining their levels in tumor samples after primary treatment could help define patients at high risk of recurrence and guide consequent courses of treatment.


Subject(s)
Ataxia Telangiectasia Mutated Proteins , Checkpoint Kinase 1 , Endometrial Neoplasms , Neoplasm Recurrence, Local , Proto-Oncogene Proteins p21(ras) , Humans , Female , Endometrial Neoplasms/genetics , Endometrial Neoplasms/pathology , Endometrial Neoplasms/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Middle Aged , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/genetics , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/metabolism , Risk Factors , Aged , ras Proteins/genetics , ras Proteins/metabolism , Gene Expression Regulation, Neoplastic , Adult , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism
7.
Cell Rep ; 43(4): 114064, 2024 Apr 23.
Article in English | MEDLINE | ID: mdl-38578830

ABSTRACT

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


Subject(s)
Basic-Leucine Zipper Transcription Factors , Checkpoint Kinase 1 , DNA-Binding Proteins , Humans , Checkpoint Kinase 1/metabolism , Phosphorylation , DNA-Binding Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , DNA Damage , Carrier Proteins/metabolism , DNA Replication , Fanconi Anemia Complementation Group Proteins/metabolism , BRCA1 Protein/metabolism , Signal Transduction , Nuclear Proteins/metabolism , Fibroblasts/metabolism , Cell Cycle Checkpoints
8.
Int J Mol Sci ; 25(8)2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38673980

ABSTRACT

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.


Subject(s)
Amidines , Checkpoint Kinase 1 , Colorectal Neoplasms , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/antagonists & inhibitors , Humans , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Colorectal Neoplasms/radiotherapy , Amidines/pharmacology , Cell Line, Tumor , Radiation, Ionizing , Radiation-Sensitizing Agents/pharmacology , DNA Replication/drug effects , Cell Proliferation/drug effects , DNA Damage/drug effects , Cell Cycle/drug effects
9.
Cancer Lett ; 592: 216898, 2024 Jun 28.
Article in English | MEDLINE | ID: mdl-38670306

ABSTRACT

Radiotherapy (RT) is used for over 50 % of cancer patients and can promote adaptive immunity against tumour antigens. However, the underlying mechanisms remain unclear. Here, we discovered that RT induces the release of irradiated tumour cell-derived microparticles (RT-MPs), which significantly upregulate MHC-I expression on the membranes of non-irradiated cells, enhancing the recognition and killing of these cells by T cells. Mechanistically, RT-MPs induce DNA double-strand breaks (DSB) in tumour cells, activating the ATM/ATR/CHK1-mediated DNA repair signalling pathway, and upregulating MHC-I expression. Inhibition of ATM/ATR/CHK1 reversed RT-MP-induced upregulation of MHC-I. Furthermore, phosphorylation of STAT1/3 following the activation of ATM/ATR/CHK1 is indispensable for the DSB-dependent upregulation of MHC-I. Therefore, our findings reveal the role of RT-MP-induced DSBs and the subsequent DNA repair signalling pathway in MHC-I expression and provide mechanistic insights into the regulation of MHC-I expression after DSBs.


Subject(s)
Ataxia Telangiectasia Mutated Proteins , Cell-Derived Microparticles , DNA Breaks, Double-Stranded , DNA Repair , Histocompatibility Antigens Class I , Signal Transduction , Up-Regulation , Humans , Cell-Derived Microparticles/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class I/genetics , Cell Line, Tumor , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/genetics , Animals , Phosphorylation , Gene Expression Regulation, Neoplastic , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , Mice , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/metabolism , Neoplasms/radiotherapy , Neoplasms/immunology
10.
Proc Natl Acad Sci U S A ; 121(18): e2322520121, 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38657044

ABSTRACT

The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1 activation. However, it is unclear how the activity of PARP1 is regulated. Here, we found that UFMylation is required for the efficient activation of CHK1 by UFMylating PARP1 at K548 during replication stress. Inactivation of UFL1, the E3 enzyme essential for UFMylation, delayed CHK1 activation and inhibits nascent DNA degradation during replication blockage as seen in PARP1-deficient cells. An in vitro study indicated that PARP1 is UFMylated at K548, which enhances its catalytic activity. Correspondingly, a PARP1 UFMylation-deficient mutant (K548R) and pathogenic mutant (F553L) compromised CHK1 activation, the restart of stalled replication forks following replication blockage, and chromosome stability. Defective PARP1 UFMylation also resulted in excessive nascent DNA degradation at stalled replication forks. Finally, we observed that PARP1 UFMylation-deficient knock-in mice exhibited increased sensitivity to replication stress caused by anticancer treatments. Thus, we demonstrate that PARP1 UFMylation promotes CHK1 activation and replication fork stability during replication stress, thus safeguarding genome integrity.


Subject(s)
Checkpoint Kinase 1 , DNA Replication , Poly (ADP-Ribose) Polymerase-1 , Animals , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/genetics , Mice , Humans , DNA Damage , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics
11.
J Biol Chem ; 300(5): 107277, 2024 May.
Article in English | MEDLINE | ID: mdl-38588804

ABSTRACT

Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase, and its dysfunction is involved in the onset of cancer and neurodegenerative disorders. PP2A functions as a trimeric holoenzyme whose composition is regulated by the methyl-esterification (methylation) of the PP2A catalytic subunit (PP2Ac). Protein phosphatase methylesterase-1 (PME-1) is the sole PP2Ac methylesterase, and the higher PME-1 expression is observed in various cancer and neurodegenerative diseases. Apart from serving as a methylesterase, PME-1 acts as a PP2A inhibitory protein, binding directly to PP2Ac and suppressing its activity. The intricate function of PME-1 hinders drug development by targeting the PME-1/PP2Ac axis. This study applied the NanoBiT system, a bioluminescence-based protein interaction assay, to elucidate the molecular mechanism that modulates unknown PME-1/PP2Ac protein-protein interaction (PPI). Compound screening identified that the CHK1 inhibitors inhibited PME-1/PP2Ac association without affecting PP2Ac methylation levels. CHK1 directly phosphorylates PP2Ac to promote PME-1 association. Phospho-mass spectrometry identified multiple phospho-sites on PP2Ac, including the Thr219, that affect PME-1 interaction. An anti-phospho-Thr219 PP2Ac antibody was generated and showed that CHK1 regulates the phosphorylation levels of this site in cells. On the contrary, in vitro phosphatase assay showed that CHK1 is the substrate of PP2A, and PME-1 hindered PP2A-mediated dephosphorylation of CHK1. Our data provides novel insights into the molecular mechanisms governing the PME-1/PP2Ac PPI and the triad relationship between PP2A, PME-1, and CHK1.


Subject(s)
Carboxylic Ester Hydrolases , Checkpoint Kinase 1 , Protein Phosphatase 2 , Protein Phosphatase 2/metabolism , Protein Phosphatase 2/genetics , Humans , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/genetics , Carboxylic Ester Hydrolases/metabolism , Carboxylic Ester Hydrolases/genetics , Phosphorylation , Luciferases/metabolism , Luciferases/genetics , Protein Binding , HEK293 Cells
12.
Eur J Med Chem ; 269: 116351, 2024 Apr 05.
Article in English | MEDLINE | ID: mdl-38547734

ABSTRACT

Checkpoint kinase 1 (CHK1) plays a crucial role in the DNA damage response pathway, making it an attractive target for cancer therapy. Herein, we present the synthesis, optimization, and evaluation of selective CHK1 inhibitors with a pyrido[3,2-d]pyrimidin-6(5H)-one scaffold. Among them, compound 11 showed single-digit nanomolar potency against CHK1 (IC50: 0.55 nM) with good kinase selectivity. Notably, 11 showed anti-proliferative effect in MV-4-11 cells singly (IC50 = 202 nM) and a synergistic effect in combination with gemcitabine in HT-29 cells (IC50 = 63.53 nM). Furthermore, the combination of 11 and gemcitabine exhibited synergistic effect in the HT-29 xenograft mouse model. Overall, this work provides a strong foundation for the development of selective CHK1 inhibitors and the therapeutic strategy for cancer.


Subject(s)
Gemcitabine , Protein Kinase Inhibitors , Humans , Mice , Animals , Checkpoint Kinase 1 , HT29 Cells , Protein Kinase Inhibitors/pharmacology , Cell Line, Tumor
13.
J Clin Invest ; 134(10)2024 Mar 26.
Article in English | MEDLINE | ID: mdl-38530355

ABSTRACT

The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.


Subject(s)
DNA Replication , Proto-Oncogene Proteins c-myc , Animals , Humans , Mice , Ataxia Telangiectasia Mutated Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Carcinogenesis/genetics , Carcinogenesis/metabolism , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/genetics , Genomic Instability , Mice, Knockout , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction , Sumoylation , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
14.
Biol Chem ; 405(6): 395-406, 2024 Jun 25.
Article in English | MEDLINE | ID: mdl-38452398

ABSTRACT

Checkpoint kinase 1 (Chk1) plays an important role in regulation of the cell cycle, DNA damage response and cell death, and represents an attractive target in anticancer therapy. Small-molecule inhibitors of Chk1 have been intensively investigated either as single agents or in combination with various chemotherapeutic drugs and they can enhance the chemosensitivity of numerous tumor types. Here we newly demonstrate that pharmacological inhibition of Chk1 using potent and selective inhibitor SCH900776, currently profiled in phase II clinical trials, significantly enhances cytotoxic effects of the combination of platinum-based drugs (cisplatin or LA-12) and TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in human prostate cancer cells. The specific role of Chk1 in the drug combination-induced cytotoxicity was confirmed by siRNA-mediated silencing of this kinase. Using RNAi-based methods we also showed the importance of Bak-dependent mitochondrial apoptotic pathway in the combined anticancer action of SCH900776, cisplatin and TRAIL. The triple drug combination-induced cytotoxicity was partially enhanced by siRNA-mediated Mcl-1 silencing. Our findings suggest that targeting Chk1 may be used as an efficient strategy for sensitization of prostate cancer cells to killing action of platinum-based chemotherapeutic drugs and TRAIL.


Subject(s)
Antineoplastic Agents , Checkpoint Kinase 1 , Cisplatin , Prostatic Neoplasms , TNF-Related Apoptosis-Inducing Ligand , Humans , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/antagonists & inhibitors , Male , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Prostatic Neoplasms/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , TNF-Related Apoptosis-Inducing Ligand/metabolism , Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Protein Kinase Inhibitors/pharmacology , Organoplatinum Compounds/pharmacology , Drug Screening Assays, Antitumor , Cell Line, Tumor , Dose-Response Relationship, Drug , Apoptosis/drug effects , Cell Proliferation/drug effects
15.
J Biol Chem ; 300(3): 105751, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38354779

ABSTRACT

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


Subject(s)
Carrier Proteins , Cell Cycle Proteins , Multiprotein Complexes , Protein Subunits , Humans , Carrier Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Checkpoint Kinase 1 , DNA/metabolism , DNA Damage , DNA Repair , Hydrophobic and Hydrophilic Interactions , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Protein Subunits/chemistry , Protein Subunits/metabolism , Protein Domains
16.
Methods Cell Biol ; 182: 221-236, 2024.
Article in English | MEDLINE | ID: mdl-38359979

ABSTRACT

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.


Subject(s)
DNA Damage , Checkpoint Kinase 1/genetics , Checkpoint Kinase 1/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Phosphorylation , Cell Cycle/genetics
17.
Pharmacol Res ; 201: 107091, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38316371

ABSTRACT

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.


Subject(s)
Antineoplastic Agents , Triterpenes , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Checkpoint Kinase 1/metabolism , Triterpenes/pharmacology , Triterpenes/therapeutic use , Tumor Suppressor Protein p53/metabolism , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Pentacyclic Triterpenes , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Ubiquitination , DNA
18.
Phytomedicine ; 126: 155177, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38412667

ABSTRACT

BACKGROUND: The mortality rate of liver cancer ranks third in the world, and hepatocellular carcinoma (HCC) is a malignant tumor of the digestive tract. Cucurbitacin B (CuB), a natural compound extracted from Cucurbitaceae spp., is the main active component of Chinese patent medicine the Cucurbitacin Tablet, which has been widely used in the treatment of various malignant tumors in clinics, especially HCC. PURPOSE: This study explored the role and mechanism of CuB in the suppression of liver cancer progression. METHODS: Cell Counting Kit-8 (CCK-8) and colony formation assays were used to detect the inhibitory function of CuB in Huh7, Hep3B, and Hepa1/6 hepatoma cells. Calcein-AM/propidium iodide (PI) staining and lactate dehydrogenase (LDH) measurement assays were performed to determine cell death. Mitochondrial membrane potential (Δψm) was measured, and flow cytometry was performed to evaluate cell apoptosis and cell cycle. Several techniques, such as proteomics, Western blotting (WB), and ribonucleic acid (RNA) interference, were utilized to explore the potential mechanism. The animal experiment was performed to verify the results of in vitro experiments. RESULTS: CuB significantly inhibited the growth of Huh7, Hep3B, and Hepa1/6 cells and triggered the cell cycle arrest in G2/M phage without leading to cell death, especially apoptosis. Knockdown of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a target of CuB, did not reverse CuB elicited cell cycle arrest. CuB enhanced phosphorylated ataxia telangiectasia mutated (p-ATM) and phosphorylated H2A histone family member X (γ-H2AX) levels. Moreover, CuB increased p53 and p21 levels and decreased cyclin-dependent kinase 1 (CDK1) expression, accompanied by improving phosphorylated checkpoint kinase 1 (p-CHK1) level and suppressing cell division cycle 25C (CDC25C) protein level. Interestingly, these phenomena were partly abolished by a deoxyribonucleic acid (DNA) protector methylproamine (MPA). Animal studies showed that CuB also significantly suppressed tumor growth in BALB/c mice bearing Hepa1/6 cells. In tumor tissues, CuB reduced the expression levels of proliferating cell nuclear antigen (PCNA) and γ-H2AX but did not change the terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) level. CONCLUSION: This study demonstrated for the first time that CuB could effectively impede HCC progression by inducing DNA damage-dependent cell cycle arrest without directly triggering cell death, such as necrosis and apoptosis. The effect was achieved through ataxia telangiectasia mutated (ATM)-dependent p53-p21-CDK1 and checkpoint kinase 1 (CHK1)-CDC25C signaling pathways. These findings indicate that CuB may be used as an anti-HCC drug, when the current findings are confirmed by independent studies and after many more clinical phase 1, 2, 3, and 4 testings have been done.


Subject(s)
Ataxia Telangiectasia , Carcinoma, Hepatocellular , Liver Neoplasms , Triterpenes , Animals , Mice , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Checkpoint Kinase 1/genetics , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/therapeutic use , Tumor Suppressor Protein p53/metabolism , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/therapeutic use , Cell Cycle Checkpoints , DNA Damage , Apoptosis , Cell Line, Tumor , Cell Proliferation
19.
OMICS ; 28(1): 8-23, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38190280

ABSTRACT

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.


Subject(s)
4-Aminopyridine/analogs & derivatives , Antineoplastic Agents , Pyrazines , Stomach Neoplasms , Humans , Cisplatin/pharmacology , Checkpoint Kinase 1/genetics , Procaterol , Stomach Neoplasms/drug therapy , Protein Kinases/genetics , Protein Kinases/metabolism , Drug Repositioning , Molecular Docking Simulation , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Drug Discovery , DNA Damage , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry
20.
Int J Mol Sci ; 25(2)2024 Jan 19.
Article in English | MEDLINE | ID: mdl-38279263

ABSTRACT

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.


Subject(s)
DNA Damage , Neoplasms , Ataxia Telangiectasia Mutated Proteins/metabolism , Checkpoint Kinase 1/genetics , Checkpoint Kinase 1/metabolism , Cell Cycle Checkpoints , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Replication , Neoplasms/drug therapy , Neoplasms/genetics
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