Checkpoint Kinase 1 Protein Research Articles

Cisplatin and Procaterol Combination in Gastric Cancer? Targeting Checkpoint Kinase 1 for Cancer Drug Discovery and Repurposing by an Integrated Computational and Experimental Approach.

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.

Checkpoint Kinase 1 (CHK1) Functions as Both a Diagnostic Marker and a Regulator of Epithelial-to-Mesenchymal Transition (EMT) in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is more difficult to treat and has a higher mortality rate than other subtypes. Although hormone receptor-targeted therapy is an effective treatment to increase survival rate in breast cancer patients, it is not suitable for TNBC patients. To address the issues, differentially expressed genes (DEGs) in TNBC patients from the Gene Expression Omnibus (GEO) database were analyzed. A total of 170 genes were obtained from three Genomic Spatial Events (GSEs) using the intersection of each GSE dataset and 61 DEGs were identified after validation with the gene enrichment analysis. We combined this with the degree scores from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein-protein interaction (PPI) network, of which 7 genes were correlated with survival rate. Finally, a proteomics database revealed that only the CHK1 protein level was differently expressed in basal-like compared with other subtypes. We demonstrated that CHK1 expression was higher in TNBC cell lines compared with non-TNBC cell lines, and CHK1 promotes epithelial to mesenchymal transition (EMT) as well as migration and invasion ability. Our study provides new insight into the TNBC subnetwork that may be useful in the prognosis and treatment of TNBC patients.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Preclinical efficacy of prexasertib in acute lymphoblastic leukemia

Preclinical efficacy of prexasertib in acute lymphoblastic leukemia

Interferon regulatory factor 1 (IRF-1) downregulates Checkpoint kinase 1 (CHK1) through miR-195 to upregulate apoptosis and PD-L1 expression in Hepatocellular carcinoma (HCC) cells.

CHK1 is considered an oncogene with overexpression in numerous cancers. However, CHK1 signalling regulation in hepatocellular carcinoma (HCC) remains unclear. CHEK1 mRNA, protein, pri-miR-195 and miR-195 expression in HCC tissue was determined by qPCR, WB and IF staining assay. Survival analyses in HCC with high- and low-CHEK1 mRNA expression was performed using TCGA database. Relative luciferase activity was investigated in HCC cells transfected with p-CHEK1 3'UTR. Apoptosis was detected by TUNEL assay. NK and CD8+ T cells were analysed by flow cytometry. CHK1 is increased in human HCC tumours compared with non-cancerous liver. High CHK1 predicts worse prognosis. IFN-γ suppresses CHK1 via IRF-1 in HCC cells. The molecular mechanism of IRF-1 suppressing CHK1 is post-transcriptional by promoting miR-195 binding to CHEK1 mRNA 3'UTR, which exerts a translational blockade. Upregulated IRF-1 inhibits CHK1, which induces apoptosis of HCC cells. Likewise, CHK1 inhibition augments cellular apoptosis in HCC tumours. This effect may be a result of increased tumour NK cell infiltration. However, IRF-1 expression or CHK1 inhibition also upregulates PD-L1 expression via increased STAT3 phosphorylation. IRF-1 induces miR-195 to suppress CHK1 protein expression. Both increased IRF-1 and decreased CHK1 upregulate cellular apoptosis and PD-L1 expression in HCC.

Human papillomavirus type 8 oncoproteins E6 and E7 cooperate in downregulation of the cellular checkpoint kinase-1.

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.

MTORC1/2 and Protein Translation Regulate Levels of CHK1 and the Sensitivity to CHK1 Inhibitors in Ewing Sarcoma Cells.

The treatment of Ewing sarcoma has changed very little in the past two decades and novel treatment approaches are needed. We recently identified that Ewing sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), the rate-limiting enzyme in the synthesis of deoxyribonucleotides. We subsequently found that the inhibition of checkpoint kinase 1 (CHK1) increases the sensitivity of Ewing sarcoma cells to inhibitors of RNR, such as gemcitabine. However, Ewing sarcoma cells exhibit high levels of the CHK1 protein, which may represent an adaptive response to elevated levels of endogenous DNA replication stress. Consequently, we began this work with the aim of determining the impact of CHK1 levels on drug sensitivity, as well as identifying the mechanisms and pathways that regulate CHK1 levels in Ewing sarcoma cells. In this report, we show that the high levels of the CHK1 protein in Ewing sarcoma cells limit the efficacy of CHK1 inhibitors. However, inhibition of mTORC1/2 activates the translational repressor 4E-BP1, reduces protein synthesis, and decreases levels of the CHK1 protein in Ewing sarcoma cells. Similarly, we identified that the CHK1 inhibitor prexasertib also activates 4E-BP1, inhibits protein synthesis, and reduces CHK1 protein levels in Ewing sarcoma cells. Moreover, the combination of prexasertib and gemcitabine was synergistic in vitro, caused tumor regression in vivo, and significantly prolonged mouse survival in a Ewing sarcoma xenograft experiment. Overall, our results provide insight into Ewing sarcoma biology and support further investigation of the CHK1 pathway as a therapeutic target in Ewing sarcoma tumors.

Abstract PR02: Exploring the effects of PARP inhibition on CHK1 activation as a potential determinant of synergy with CHK1 inhibition

Abstract PARP inhibitors (PARPi) have improved the outcomes of patients with high-grade serous ovarian carcinomas (HGSOC) whose tumors harbor mutations in homologous recombination genes such BRCA1 and BRCA2. However, only 20% of cases with this aggressive subtype of ovarian cancer benefit from PARPi, thus highlighting the essential need to identify novel therapeutic strategies for the remainder of these patients. We have recently reported that off-target effects vary among two PARP inhibitors with different trapping potencies. The FDA-approved PARPi, Olaparib, activated the checkpoint kinase 1 (CHK1) protein at serine 345 and arrested cells in the G2/M phase; however, these effects were not observed after treatment with Veliparib, the least potent PARPi measured by the PARP trapping ability. In this study, we treated HGSOC cell lines with the five most clinically advanced PARP inhibitors and investigated the effect of varying PARP trapping potencies on the expression levels of major proteins that are involved in cell cycle pathways via Western blot and cell cycle analysis. The synergy between the most potent (Talazoparib) or least potent PARP trappers and a CHK1 inhibitor was also explored in HGSOC patient-derived xenograft (PDX) models. We hypothesize that PARP inhibitors that do not have an effect on the cell cycle will have greater synergy with CHK1 inhibitors. Our results reveal that the four most potent PARP trappers—olaparib, rucaparib, niraparib, and talazoparib--activate CHK1 protein and decrease total protein levels of CHK1’s downstream substrate CDC25C. However, we did not observe any significant changes in expression levels in these two proteins after treatment with veliparib. We also observed an arrest in the S/G2 phase of the cell cycle after treatment with the four most potent PARP inhibitors. Finally, the growth of our PDX model was significantly inhibited by combined treatment with veliparib and the CHK1 inhibitor MK-8776 compared to either single-agent therapy or talazoparib combined with MK-8776. These results suggest that there is a distinct off-target effect that differs among the least and most potent PARP inhibitors, with veliparib being more synergistic with CHK1 inhibition. This study will help to expand the use of PARP inhibitors in combination with CHK1 inhibitors and emphasizes the importance of single-agent off-target effects to develop optimal combination therapies. This abstract is also being presented as Poster A11. Citation Format: Monicka Wielgos-Bonvallet, Paulina Cybulska, Elke Van Oudenhove, Petar Jelinic, Douglas A. Levine. Exploring the effects of PARP inhibition on CHK1 activation as a potential determinant of synergy with CHK1 inhibition. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr PR02.

Abstract 4149: Inhibition of ribonucleotide reductase (RNR) in Ewing sarcoma cells results in suppression of protein synthesis

Abstract Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. EWS-FLI1 is required for tumor growth and survival. However, directly targeting EWS-FLI1 with drugs has been challenging and an alternative therapeutic approach is to identify unique vulnerabilities incurred by the oncoprotein. In previous work, we identified that Ewing sarcoma cells are uniquely vulnerable to drugs that inhibit ribonucleotide reductase (RNR), which is the rate limiting enzyme in the synthesis of deoxyribonucleotides. Furthermore, we also showed that blocking the function of checkpoint kinase 1 (CHK1), a major regulator of the response to impaired DNA replication, using either a small molecule inhibitor or siRNA-mediated knockdown causes synergistic toxicity with gemcitabine, an irreversible inhibitor of the RRM1 subunit of RNR, in Ewing sarcoma cells. In the current work, we identifiy that inhibition of RNR in Ewing sarcoma cells in vitro and in vivo results in the suppression of protein synthesis. Moreover, we have found that the treatment of Ewing sarcoma cell lines with gemcitabine results in an increase in the active (unphosphorylated) form of 4E-BP1, which is a well-described inhibitor of protein translation. However, in contrast to the Ewing sarcoma cells, treatment of other cell types with gemcitabine did not alter 4E-BP1 phosphorylation or protein synthesis. Currently, ongoing work is focused on the in vivo testing of gemcitabine, alone and in combination with CHK1, ataxia telangiectasia and rad3-related protein (ATR), and protein synthesis inhibitors, as a novel therapeutic approach for the treatment of Ewing sarcoma. Citation Format: Stacia Koppenhafer, Kelli Goss, David Gordon. Inhibition of ribonucleotide reductase (RNR) in Ewing sarcoma cells results in suppression of protein synthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4149.

Transformer2 proteins protect breast cancer cells from accumulating replication stress by ensuring productive splicing of checkpoint kinase 1

Increased expression levels of the RNA splicing regulator Transformer2β (abbreviated Tra2β) have been reported in several types of cancer. Recent work has revealed an intimate cross-regulation between Tra2β and the highly similar Tra2α protein in human breast cancer cells, though these two proteins are encoded by separate genes created by a gene duplication that occurred over 500 million years ago. This cross-regulation involves splicing control of a special class of exons, called poison exons. Down-regulation of Tra2β reduces splicing inclusion of a poison exon in the mRNA encoding Tra2α, thereby up-regulating Tra2α protein expression. This buffers any splicing changes that might be caused by individual depletion of Tra2β alone. Discovery of this cross-regulation pathway, and its by-pass by joint depletion of both human Tra2 proteins, revealed Tra2 proteins are essential for breast cancer cell viability, and led to the identification of important targets for splicing control. These exons include a critical exon within the checkpoint kinase 1 (CHK1) gene that plays a crucial function in the protection of cancer cells from replication stress. Breast cancer cells depleted for Tra2 proteins have reduced CHK1 protein levels and accumulate DNA damage. These data suggest Tra2 proteins and/or their splicing targets as possible cancer drug targets.

Checkmate to CHK1 in T-cell ALL?

DNA replication ensures accurate duplication of the original genetic information present in a cell in order for it to be properly transmitted to daughter cells. However, replication can be perturbed, for instance in rapidly dividing cancer cells, in a process referred to as replication stress (RS). Checkpoint kinase 1 (CHK1) is an essential component of the ATR-dependent DNA damage-response pathway that protect cells from RS by preventing replication fork collapse and activating homologous DNA repair. The ATR-CHK1 pathway is triggered upon exposure of single-stranded DNA that arises with the stalling of replication forks [1], and it is required to reset proper origin firing, and to promote fork stability and checkpoint activation, delaying mitosis until replication is completed and thereby avoiding mitotic catastrophe [2]. Whereas these functions point towards a tumor suppressor role for CHK1, mouse models modulating ATR-CHK1 expression and genetic evidence from human tumors suggest otherwise: Atr and Chk1 knock-out models do not display higher tumor frequency; Chk1 favors oncogene-induced transformation in mice; CHK1 is frequently overexpressed in human cancers, while loss-of-function mutations are rare [2, 3]. Moreover, CHK1 affords protection against DNA damaging agents, a fact that prompted the use of CHK1 inhibitors as chemosensitizers [4]. Similarly, tumors whose oncogenic profile fuels RS were proposed to become addicted to ATR-CHK1 response [1, 2]. In our recent study published in Oncogene [5], we hypothesized that T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological cancer arising from T-cell precursor clonal expansion, could be one of such tumors and showed that CHK1 plays a key role in T-ALL cell maintenance. T-ALL cells tend to be highly proliferative due to a myriad of genetic lesions that culminate in cyclin-dependent kinase hyperactivation, and deregulated progression of S-phase that may impact on DNA replication [5, 6]. We found that T-ALL cells overexpressed CHK1 mRNA and protein as compared to normal hematopoietic progenitors. This was accompanied by aberrantly high CHK1 kinase activity, likely triggered by high basal levels of RS [5]. Experimental inactivation of CHK1, by a CHK1 selective inhibitor (PF-00477736) or by gene silencing, demonstrated that CHK1 is essential to control the accumulation of RS and to prevent apoptosis of T-ALL cells that appear to enter mitosis without having concluded DNA replication. Furthermore, accumulation of DNA damage in the context of CHK1 loss induced the activation of the ATM-CHK2 DNA double-strand break (DSB) response pathway, likely due to DSB formation upon the collapse of stalled replication forks. T-ALL apoptosis upon CHK1 inactivation was in the first instance dependent on ATM and caspase-3, since ATM inhibition prevented caspase-3 cleavage and rescued T-ALL cell viability despite sustained elevated amounts of RS markers [5]. Following the demonstration that T-ALL cells were eliminated using a CHK1 small molecule inhibitor as single agent, we showed that this effect was leukemia-specific, since normal T-cell progenitors were not sensitive to the low doses of PF-00477736 that killed primary T-ALL patient cells. Moreover, the in vitro anti-leukemia effect of PF-00477736 was not prevented by microenvironment pro-survival factors, and the potential clinical value of CHK1 inhibition was further demonstrated by the fact that PF-00477736 limited the growth of xenografted T-ALL tumors [5]. Interestingly, our preliminary analyses indicated that T-ALL cells expressing higher CHK1 levels appeared more sensitive to CHK1 pharmacological inhibition, suggesting that CHK1 expression could be a suitable drug response marker in T-ALL patients. As clinical trials against ATR-CHK1 pathway may be envisaged, this issue warrants extended T-ALL patient analysis. T-ALL constitutes only a fraction of all ALL cases, but it associates with high-risk. Therapeutic options with less detrimental side-effects and/or effective upon relapse are most desired. Our findings defining CHK1 as a ‘subverted’ tumor suppressor that stands in T-ALL as a major guardian of leukemia cell survival, thereby formally acting as an oncogene, reinforce a new way of viewing the mechanisms of cancer progression [2] and may set the ground for anti-leukemia breakthrough approaches. In this context, it is important to understand the mechanisms of CHK1 upregulation in T-ALL. We thoroughly documented CHK1 transcript overexpression in primary T-ALL [5]. However, how this occurs remains undetermined. Maybe transcription factors known to activate CHK1, such as E2F (downstream of G1/S-phase CDK activity) or MYC (downstream of NOTCH1), are involved in CHK1 overexpression in T-ALL. Or maybe as yet unidentified CHK1 regulatory elements are mutated or epigenetically altered. Curiously, in contrast to our findings, Chk1 mRNA downregulation was documented in a murine T-ALL model [7]. A more integrative view of the role of CHK1 in T-ALL is therefore required. We believe CHK1 downregulation may occur at T-ALL initiation, driving genomic instability secondary to an increase in RS. As the pro-proliferative oncogenic program establishes and RS rises, leukemic cells are naturally selected for their ability to upregulate CHK1 as a means to maintain RS levels compatible with cell viability.

Abstract 496: Checkpoint kinase 1 inhibition suppresses cell growth and enhances cisplatin sensitivity in medulloblastoma cells

Abstract Background: Medulloblastoma is the most common malignant brain tumor in children and remains a therapeutic challenge due to its significant therapy-related morbidity. Checkpoint kinase 1 (CHK1) is highly expressed in many cancers and regulates critical steps in mitotic progression and DNA-damage response. Activation of CHK1 pathway promotes treatment resistance in tumor cells. Recent studies suggest that targeting CHK1 with a small molecule inhibitor, to sensitize tumors to a variety of DNA-damaging agents, is a promising approach to tumor therapy. Methods: The expression of CHK1 mRNA in medulloblastoma patient samples and cell lines were examined using microarray analysis and qRT-PCR respectively. Western blot analysis was conducted on medulloblastoma cell lines to analyze expression level of CHK1 protein. The effect of AZD7762, a small molecule inhibitor of CHK1, on colony forming ability of medulloblastoma cell lines in combination with cisplatin was examined using colony formation assay and methylcellulose assay. The impact of AZD7762 in combination with cisplatin on tumor cell proliferation was further evaluated in real-time using xCELLigence. The effect of AZD7762 on cell viability was assessed using Guava ViaCount assay. Furthermore, the effects on key signaling pathways were analyzed using immunofluorescence, senescence assay, and apoptosis assay. Results: Analysis of gene expression and western blot experiments revealed that CHK1 mRNA and protein levels are over expressed in all medulloblastoma patient samples and in cell lines when compared to normal pediatric cerebellum. High CHK1 expression correlates with adverse outcomes in a large cohort of medulloblastoma patients. Inhibition of CHK1 by a low nanomolar concentration of AZD7762 potently inhibited cell growth, suppressed the colony-forming ability, and increased cellular apoptosis of medulloblastoma cells. Furthermore, AZD7762 pretreatment sensitized medulloblastoma cells to cisplatin synergistically. Conclusions: CHK1 expression is a prognostic marker in medulloblastoma and targeting CHK1 with a small molecule inhibitor, in combination with cisplatin, is an attractive strategy in treatment of medulloblastoma that warrants further investigation. Citation Format: Monil Shah, Eric Prince, Sujatha Venkataraman, Ilango Balakrishnan, Irina Alimova, Peter Harris, Marc Remeke, Michael D. Taylor, Michael H. Handler, Nicholas K. Foreman, Rajeev Vibhakar. Checkpoint kinase 1 inhibition suppresses cell growth and enhances cisplatin sensitivity in medulloblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 496. doi:10.1158/1538-7445.AM2015-496

MB-22 * CHECKPOINT KINASE 1 EXPRESSION IS AN ADVERSE PROGNOSTIC MARKER AND THERAPEUTIC TARGET IN MYC-DRIVEN MEDULLOBLASTOMA

BACKGROUND: Medulloblastoma is the most common childhood malignant intracranial tumor that carries significant therapy-related morbidity. Checkpoint kinase 1 (CHK1) is an integral component of the cell cycle as well as the DNA Damage Response (DDR) pathway, Previous work has demonstrated the effectiveness of inhibiting CHK1 with small-molecule inhibitors, such as AZD-7762, in epithelial tumors, but research is scarce in terms of targeting the DDR for treatment in CNS malignancies. METHODS: Low-dose administration of AZD-7762 and cisplatin, the standard chemotherapy agent for medulloblastoma, were used both separately and together to assess the effect of CHK1 inhibition on c-Myc amplified medulloblastoma cell lines. Clonogenic assays were performed to assess the effect of AZD-7762, with and without cisplatin, on cell survival. Flow cytometric cell cycle and γH2AX analyses were utilized to investigate the impact of AZD-7762 on cell status and active apoptosis, respectively. RESULTS: Genetic analysis revealed a significant increase in expression of CHK1 in medulloblastoma which correlated with previously established data sets. Increased CHK1 mRNA and protein expression in medulloblastoma cell lines versus normal cerebellum was confirmed via qPCR and western blot assays. Targeting CHK1 with low-dose AZD-7762 resulted in a decrease in cell viability and cell survival as well as an increase in actively apoptotic cells. Finally, combined treatment of low-dose AZD-7762 (20 nM) with low-dose cisplatin (175 nM) was synergistic towards reducing cell survival. CONCLUSION: Overall, results demonstrate that small-molecule inhibition of CHK1 alongside the ubiquitous chemotherapeutic, cisplatin, is more advantageous than either treatment alone, especially for Group 3 medulloblastoma, and therefore this combined therapeutic approach serves as an avenue for further investigation.

Checkpoint kinase1 (CHK1) is an important biomarker in breast cancer having a role in chemotherapy response.

Background:Checkpoint kinase1 (CHK1), which is a key component of DNA-damage-activated checkpoint signalling response, may have a role in breast cancer (BC) pathogenesis and influence response to chemotherapy. This study investigated the clinicopathological significance of phosphorylated CHK1 (pCHK1) protein in BC.Method:pCHK1 protein expression was assessed using immunohistochemistry in a large, well-characterized annotated series of early-stage primary operable invasive BC prepared as tissue microarray (n=1200).Result:pCHK1 showed nuclear and/or cytoplasmic expression. Tumours with nuclear expression showed positive associations with favourable prognostic features such as lower grade, lower mitotic activity, expression of hormone receptor and lack of expression of KI67 and PI3K (P<0.001). On the other hand, cytoplasmic expression was associated with features of poor prognosis such as higher grade, triple-negative phenotype and expression of KI67, p53, AKT and PI3K. pCHK1 expression showed an association with DNA damage response (ATM, RAD51, BRCA1, KU70/KU80, DNA-PKCα and BARD1) and sumoylation (UBC9 and PIASγ) biomarkers. Subcellular localisation of pCHK1 was associated with the expression of the nuclear transport protein KPNA2. Positive nuclear expression predicted better survival outcome in patients who did not receive chemotherapy in the whole series and in ER-positive tumours. In ER-negative and triple-negative subgroups, nuclear pCHK1 predicted shorter survival in patients who received cyclophosphamide, methotrexate and 5-florouracil chemotherapy.Conclusions:Our data suggest that pCHK1 may have prognostic and predictive significance in BC. Subcellular localisation of pCHK1 protein is related to its function.

Checkpoint kinase 1 protein expression indicates sensitization to therapy by checkpoint kinase 1 inhibition in non–small cell lung cancer

BackgroundWhen presenting with advanced stage disease, lung cancer patients have <5% 5-y survival. The overexpression of checkpoint kinase 1 (CHK1) is associated with poorer outcomes and may contribute to therapy resistance. Targeting CHK1 with small-molecule inhibitors in p53 mutant tumors might improve the effectiveness of chemotherapy and radiotherapy in non–small cell lung cancer (NSCLC). MethodsWe evaluated CHK1 messenger RNA and protein levels in multiple NSCLC cell lines. We assessed cell line sensitization to gemcitabine, pemetrexed, and radiotherapy by CHK1 inhibition with the small molecule AZD7762 using proliferation and clonogenic cell survival assays. We analyzed CHK1 signaling by Western blotting to confirm that AZD7762 inhibits CHK1. ResultsWe selected two p53 mutant NSCLC cell lines with either high (H1299) or low (H1993) CHK1 levels for further analysis. We found that AZD7762 sensitized both cell lines to gemcitabine, pemetrexed, and radiotherapy. Chemosensitization levels were greater, however, for the higher CHK1 protein expressing cell line, H1299, when compared with H1993. Furthermore, analysis of the CHK1 signaling pathway showed that H1299 cells have an increased dependence on the CHK1 pathway in response to chemotherapy. There was no increased sensitization to radiation in H1299 versus H1993. ConclusionsCHK1 inhibition by AZD7762 preferentially sensitizes high CHK1 expressing cells, H1299, to anti-metabolite chemotherapy as compared with low CHK1 expressing H1993 cells. Thus, CHK1 inhibitors may improve the efficacy of standard lung cancer therapies, especially for those subgroups of tumors harboring higher expression levels of CHK1 protein.

CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells

ObjectiveOverexpression of checkpoint kinase 1 (CHK1) is associated with poorer patient outcome and therapeutic resistance in multiple tumor models. Inhibition of CHK1 has been proposed as a strategy to increase the effectiveness of chemotherapeutic agents, especially in p53-deficient tumors. In this study, we evaluated the effects of a novel CHK1 inhibitor, MK-8776, in combination with pemetrexed (PMX) on cell proliferation and survival in a panel of p53 mutant non-small cell lung cancer (NSCLC) cell lines. MethodsWe examined CHK1 expression in 442 resected lung adenocarcinoma specimens using Affymetrix U133A gene expression arrays. We correlated CHK1 mRNA expression with patient survival, tumor differentiation and genomic complexity. We evaluated CHK1 levels in NSCLC cell lines and identified four p53 mutant cell lines with variable CHK1 expression (H1993, H23, H1437 and H1299) based on publicly available gene expression data. We confirmed differential CHK1 mRNA and CHK1 protein levels by qRT-PCR, ELISA, Western Blot analysis (WB) and immunohistochemistry. We examined cell line sensitization to PMX in response to CHK1 inhibition with MK-8776 using WST-1 and clonogenic survival assays. ResultsWe found that elevated CHK1 expression in primary lung adenocarcinomas correlates with poor tumor differentiation and significantly worse patient survival. Tumors with elevated CHK1 mRNA levels have a higher number of gene mutations and DNA copy number gain or amplifications. CHK1 inhibition by MK-8776 enhances sensitivity of NSCLC cell lines to PMX. CHK1 mRNA and protein expression are variable among NSCLC cell lines, and cells expressing higher levels of CHK1 protein are more sensitive to the CHK1 inhibition by MK-8776 as compared to low CHK1 expressing cells. ConclusionsThese findings suggest that CHK1 levels may not only serve as a biomarker of poor prognosis in surgically-resected lung adenocarcinomas, but could also be a predictive marker for CHK1 inhibitor sensitivity, pending in vivo and clinical confirmation.

Abstract B22: Combined disruption of ATM and CHK1 functionalities reveals redundancies in the DNA damage response pathways and results in synthetic growth inhibition following γ-irradiation

Abstract Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints, and promoting DNA damage repair. The phosphoinositide 3-kinase related kinases (PIKKs) Ataxia Telangiectasia-Mutated (ATM), ATM and Rad 3-related kinase (ATR), and DNA-dependent Protein Kinase (DNA-PK) are crucial for lesion sensing and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anti-cancer regimens. We hypothesized that ATM-deficient cells depend on signaling through CHK1 for survival after genotoxic exposures. The purpose of this study was to develop isogenic cell lines that are stably depleted for CHK1, ATM, or both and examine cross-talk and compensatory effects on cellular growth in general and the G2/M checkpoint in particular following treatment with γ-irradiation (IR). Human epithelial cell cultures (HME-CC) were stably transduced with either control or CHK1-targeting lentiviral shRNA particles in a wild-type or ATM-deficient background. Cell line validation and characterization was performed with cellular assays in conjunction with real-time quantitative PCR and Western blotting. Cell cycle profiling was done by flow cytometry. In addition, pharmacological approaches were used to functionally disrupt the proteins of interest for this study. We show that individual depletion of CHK1 and ATM results in augmented cellular proliferation as reflected in shortened cell population doubling times. Notably, a high level of depletion of CHK1 (HEK293 and HME-CC cells) or ATM (HME-CC) renders cells radiosensitive without abrogating their IR-mediated G2/M checkpoint arrest. Our data indicates that following IR, CHK1-deficient cells display enhanced ATM phosphorylation compared to their wild-type counterparts and conversely, ATM-depleted cells exhibit enhanced CHK1 phosphorylation, thus revealing a redundancy in the G2/M checkpoint enforcement after DNA damage. We provide evidence that this ATM-CHK1 cooperation is mediated at least in part by a feedback regulatory loop with the phosphatase PP2A. CHK1-depleted cells exhibit a lower nuclear abundance of the PP2A catalytic subunit, and a concurrent increase in the inhibitory phosphorylated state of the PP2A catalytic subunit compared to CHK1-proficient cells. Intriguingly, stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels, suggesting that loss of CHK1 by ATM-deficient cells may be synthetically lethal. Combined ATM depletion (10% of wild-type level) and CHK1-deficiency (50%) resulted in an additive attenuation of the G2/M checkpoint response compared to the individual knockdown, whereas pharmacological ATM inhibition in conjunction with 90% CHK1 depletion abrogated the early G2/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time-points. Furthermore, at 48 h post-IR the individually-depleted cell lines continued to proliferate, whereas the double-deficient cell line ATM-CHK1 halted proliferation. We conclude that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage, abrogates the checkpoint arrest, and leads to synthetic growth inhibition. A combined targeting of ATM and CHK1 functionalities merits further investigation for developing more efficient anti-neoplastic treatments. Citation Format: Stela S. Palii, Richard S. Paules. Combined disruption of ATM and CHK1 functionalities reveals redundancies in the DNA damage response pathways and results in synthetic growth inhibition following γ-irradiation. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B22.

Dissecting cellular responses to irradiation via targeted disruptions of the ATM-CHK1-PP2A circuit

Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (ATM), ATM and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, ATM or both for dissecting cross-talk and compensatory effects on G₂/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G₂/M checkpoint arrest. ATM phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G₂/M checkpoint response compared with the individual knockdowns. ATM inhibition and 90% CHK1 depletion abrogated the early G₂/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments.

Ciprofloxacin‐mediated cell proliferation inhibition and G2/M cell cycle arrest in rat tendon cells

To investigate the effect of ciprofloxacin on the proliferation and cell cycle progression of tendon cells, and to explore the potential molecular mechanism of ciprofloxacin-associated tendinopathy by analyzing the expression of cell cycle-related cyclin and cyclin-dependent kinase (CDK). Rat Achilles tendon cells were treated with ciprofloxacin and then assessed by MTT assay, flow cytometric analysis, and fluorescence confocal microscopy. Levels of messenger RNA (mRNA) for CDK-1 and cyclin B were determined by reverse transcriptase-polymerase chain reaction. Protein expression of CDK-1, cyclin B, checkpoint kinase 1 (CHK-1), and polo-like kinase 1 (PLK-1) was determined by Western blot analysis. Ciprofloxacin inhibited tendon cell proliferation and caused cell cycle arrest at the G2/M phase. Confocal microscopy revealed that chromosomes in ciprofloxacin-treated cells neither properly aligned along the equatorial planes nor segregated successfully during metaphase. Mitotic arrest, misaligned chromosomes, and poor bipolar spindle formation were observed in ciprofloxacin-treated cells. CDK-1 and cyclin B protein and mRNA were both down-regulated. CHK-1 protein expression was also suppressed, but PLK-1 protein expression was up-regulated by ciprofloxacin. Our findings suggest a possible mechanism of ciprofloxacin-associated tendinopathy. Down-regulation of CHK-1 and up-regulation of PLK-1 may account for mitotic arrest observed in ciprofloxacin-treated cells.

Checkpoint kinase 1 (CHK1) protein and mRNA expression is downregulated in aggressive variants of human lymphoid neoplasms

CHK1: gene encodes for a serine/threonine kinase involved in the regulation of cell cycle progression and DNA damage checkpoints. To determine the role of CHK1 in the pathogenesis of lymphoid neoplasms and its relationship to other DNA damage response genes, we have analyzed the gene status, protein, and mRNA expression in a series of tumors and nonneoplastic lymphoid tissues. CHK1 protein and mRNA expression levels were very low in both reactive tissues and resting lymphoid cells, whereas tumor samples showed a variable pattern of expression related to their proliferative activity. However, seven aggressive tumors showed a dissociate pattern of extremely low or negative protein expression in spite of a high proliferative activity. Four of these tumors were diffuse large B-cell lymphomas (DLCLs) with concordant reduced levels of mRNA, whereas one blastoid mantle cell lymphoma (B-MCL) and two DLCLs had relatively normal levels of mRNA. No gene mutations, deletions, or hypermethylation of the promoter region were detected in any of these cases. In all these tumors ATM, CHK2, and p53 genes were wild type. These findings suggest that CHK1 inactivation in NHLs occurs by loss of protein expression in a subset of aggressive variants alternatively to ATM, CHK2, and p53 alterations.