Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe.

Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)- and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive single-strand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells.

RecQL4 tethering on the pre-replicative complex induces unscheduled origin activation and replication stress in human cells.

Sequential activation of DNA replication origins is precisely programmed and critical to maintaining genome stability. RecQL4, a member of the conserved RecQ family of helicases, plays an essential role in the initiation of DNA replication in mammalian cells. Here, we showed that RecQL4 protein tethered on the pre-replicative complex (pre-RC) induces early activation of late replicating origins during S phase. Tethering of RecQL4 or its N terminus on pre-RCs via fusion with Orc4 protein resulted in the recruitment of essential initiation factors, such as Mcm10, And-1, Cdc45, and GINS, increasing nascent DNA synthesis in late replicating origins during early S phase. In this origin activation process, tethered RecQL4 was able to recruit Cdc45 even in the absence of cyclin-dependent kinase (CDK) activity, whereas CDK phosphorylation of RecQL4 N terminus was required for interaction with and origin recruitment of And-1 and GINS. In addition, forced activation of replication origins by RecQL4 tethering resulted in increased replication stress and the accumulation of ssDNAs, which can be recovered by transcription inhibition. Collectively, these results suggest that recruitment of RecQL4 to replication origins is an important step for temporal activation of replication origins during S phase. Further, perturbation of replication timing control by unscheduled origin activation significantly induces replication stress, which is mostly caused by transcription-replication conflicts.

Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes.

In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes "replicative stress" that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair.

Cell division cycle 7 kinase is a negative regulator of cell-mediated collagen degradation.

Although extensive work has delineated many of the mechanisms of extracellular matrix (ECM) production, far less is known about pathways that regulate ECM degradation. This is particularly true of cellular internalization and degradation of matrix, which play an underappreciated role in ECM metabolism and lung fibrosis. For example, genetic perturbation of this pathway leads to exacerbated fibrosis in experimental animal models. In this work, we present the results of an unbiased screen of Drosophila phagocytes that yielded multiple genes that, when silenced, led to increased collagen uptake. We further describe the function of cell division cycle 7 kinase (CDC7) as a specific suppressor of collagen uptake. We show that the genetic or pharmacological inhibition of CDC7 results in increased expression of the collagen endocytic receptor Endo180. Chromobox 5 (CBX5) is a putative target of CDC7, and genetic silencing of CBX5 also results in increased Endo180 and collagen uptake. Finally, CRISPR-mediated activation of Endo180 expression results in increased collagen uptake, suggesting that CDC7 regulates collagen internalization through increased Endo180 expression. Targeting the regulatory elements of the collagen degradative machinery may be a useful therapeutic approach in diseases of fibrosis or malignancy.

Identification of a new class of potent Cdc7 inhibitors designed by putative pharmacophore model: Synthesis and biological evaluation of 2,3-dihydrothieno[3,2-d]pyrimidin-4(1H)-ones.

Cell division cycle 7 (Cdc7) is a serine/threonine kinase that plays important roles in the regulation of DNA replication process. A genetic study indicates that Cdc7 inhibition can induce selective tumor-cell death in a p53-dependent manner, suggesting that Cdc7 is an attractive target for the treatment of cancers. In order to identify a new class of potent Cdc7 inhibitors, we generated a putative pharmacophore model based on in silico docking analysis of a known inhibitor with Cdc7 homology model. The pharmacophore model provided a minimum structural motif of Cdc7 inhibitor, by which preliminary medicinal chemistry efforts identified a dihydrothieno[3,2-d]-pyrimidin-4(1H)-one scaffold having a heteroaromatic hinge-binding moiety. The structure-activity relationship (SAR) studies resulted in the discovery of new, potent, and selective Cdc7 inhibitors 14a, c, e. Furthermore, the high selectivity of 14c, e for Cdc7 over Rho-associated protein kinase 1 (ROCK1) is discussed by utilizing a docking study with Cdc7 and ROCK2 crystal structures.

2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones bearing 3-methylpyrazole hinge binding moiety: Highly potent, selective, and time-dependent inhibitors of Cdc7 kinase.

In order to increase the success rate for developing new Cdc7 inhibitors for cancer therapy, we explored a new chemotype which can comply with the previously-constructed pharmacophore model. Substitution of a pyridine ring of a serendipitously-identified Cdc7 inhibitor 2b with a 3-methylpyrazole resulted in a 4-fold increase in potency and acceptable kinase selectivity, leading to the identification of thieno[3,2-d]pyrimidin-4(3H)-one as an alternative scaffold. Structure-activity relationship (SAR) study revealed that incorporation of a substituted aminomethyl group into the 2-position improved kinase selectivity. Indeed, a pyrrolidinylmethyl derivative 10c was a potent Cdc7 inhibitor (IC50=0.70nM) with high selectivity (Cdk2/Cdc7≥14,000, ROCK1/Cdc7=200). It should be noted that 10c exhibited significant time-dependent Cdc7 inhibition with slow dissociation kinetics, cellular pharmacodynamic (PD) effects, and COLO205 growth inhibition. Additionally, molecular basis of high kinase selectivity of 10c is discussed by using the protein structures of Cdc7 and Cdk2.

The Replication Initiation Protein Sld3/Treslin Orchestrates the Assembly of the Replication Fork Helicase during S Phase.

The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2-7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2-7, whereas expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2-7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.

Cell division cycle 7-kinase inhibitor PHA-767491 hydrochloride suppresses glioblastoma growth and invasiveness.

BACKGROUND: Genomic instability is a hallmark of cancer cells, and this cellular phenomenon can emerge as a result of replicative stress. It is possible to take advantage of replicative stress, and enhance it in a targeted way to fight cancer cells. One of such strategies involves targeting the cell division cycle 7-related protein kinase (CDC7), a protein with key roles in regulation of initiation of DNA replication. CDC7 overexpression is present in different cancers, and small molecule inhibitors of the CDC7 have well-documented anti-tumor effects. Here, we aimed to test the potential of CDC7 inhibition as a new strategy for glioblastoma treatment. METHODS: PHA-767491 hydrochloride was used as the CDC7 inhibitor. Two glioblastoma cell lines (U87-MG and U251-MG) and a control cell line (3T3) were used to characterize the effects of CDC7 inhibition. The effect of CDC7 inhibition on cell viability, cell proliferation, apoptosis, migration, and invasion were analyzed. In addition, real-time PCR arrays were used to identify the differentially expressed genes in response to CDC7 inhibition. RESULTS: Our results showed that CDC7 inhibition reduces glioblastoma cell viability, suppresses cell proliferation, and triggers apoptosis in glioblastoma cell lines. In addition, we determined that CDC7 inhibition also suppresses glioblastoma cell migration and invasion. To identify molecular targets of CDC7 inhibition, we used real-time PCR arrays, which showed dysregulation of several mRNAs and miRNAs. CONCLUSIONS: Taken together, our findings suggest that CDC7 inhibition is a promising strategy for treatment of glioblastoma.

XL413, a cell division cycle 7 kinase inhibitor enhanced the anti-fibrotic effect of pirfenidone on TGF-ß1-stimulated C3H10T1/2 cells via Smad2/4.

Pirfenidone is an orally bioavailable synthetic compound with therapeutic potential for idiopathic pulmonary fibrosis. It is thought to act through antioxidant and anti-fibrotic pathways. Pirfenidone inhibits proliferation and/or myofibroblast differentiation of a wide range of cell types, however, little studies have analyzed the effect of pirfenidone on the mesenchymal stem cells, which play an important role on the origin of myofibroblasts. We recently found that pirfenidone had anti-proliferative activity via G1 phase arrest and cell division cycle 7 (Cdc7) kinase expression decrease in transforming growth factor-ß1 (TGF-ß1)-stimulated murine mesenchymal stem C3H10T1/2 cells. Pirfenidone also had inhibiting effect on the migration and α-SMA expression. Moreover, in this study we showed for the first time that Cdc7 inhibitor XL413 enhanced the anti-fibrotic activity of pirfenidone via depressed the expression of Smad2/4 proteins, and also prevented the nuclear accumulation and translocation of Smad2 protein. In conclusion, we demonstrated that pirfenidone inhibited proliferation, migration and differentiation of TGF-ß1-stimulated C3H10T1/2 cells, which could be enhanced by Cdc7 inhibitor XL413, via Smad2/4. Combination with pirfenidone and XL413 might provide a potential candidate for the treatment of TGF-ß1 associated fibrosis. It needs in vivo studies to further validate its therapeutic function and safety in the future.

Cell division cycle 7 mediates transforming growth factor-ß-induced smooth muscle maturation through activation of myocardin gene transcription.

Smooth muscle (SM) development consists of several processes, including cell fate determination, differentiation, and maturation. The molecular mechanisms controlling SM early differentiation have been studied extensively. However, little is known about the mechanism underlying SM maturation. Cell division cycle 7 (Cdc7) has been shown to regulate cell fate determination in the initial phase of transforming growth factor-ß (TGF-ß)-induced SM differentiation. Our present study indicates that Cdc7 also regulates SM maturation. Knockdown of Cdc7 suppresses TGF-ß-induced expression of SM myosin heavy chain, a late marker of SM differentiation. Cdc7 overexpression, on the other hand, enhances SM myosin heavy chain expression. Interestingly, Cdc7 activates the mRNA expression and promoter activity of myocardin (Myocd), a master regulator of SM differentiation, whose transcription is blocked in the initial phase of the differentiation because TGF-ß does not induce Myocd mRNA until after the early SM markers are induced. These data suggest that Cdc7 mediates TGF-ß-induced SM maturation via activation of Myocd transcription. Mechanistically, Cdc7 physically and functionally interacts with Nkx2.5 to regulate Myocd promoter activity. Cdc7 appears to enhance Nkx2.5 binding to Myocd promoter, leading to Myocd activation. Taken together, our studies demonstrate that Cdc7 regulates the initial and late phase of SM differentiation through distinct mechanisms.

Assessment of Effects of Investigational TAK-931, an Oral Cell Division Cycle 7 Kinase Inhibitor on the QTc Intervals in Patients With Advanced Solid Tumors.

TAK-931, a novel, selective, small-molecule inhibitor of cell division cycle 7 has been investigated in multiple clinical trials in patients with advanced solid tumors. An integrated analysis using data from 2 clinical studies assessed effects of TAK-931 on electrocardiogram QT intervals and heart rate (HR). Pharmacokinetic samples and matched triplicate electrocardiogram data were collected in 48 patients with cancer receiving oral administration of TAK-931 50 or 80 mg once daily. The relationships between TAK-931 plasma concentrations and the HR-corrected QT interval via Fridericia (QTcF) or population (QTcP) and HR were analyzed using linear mixed-effects models with fixed effects for day and time. At the geometric mean maximum TAK-931 plasma concentrations after administration of 50 mg, an HR change of 3.40 beats per minute (90%CI, 1.86-4.80) was predicted. Change in QTcF of -3.41 milliseconds (90%CI, -5.77 to -1.17) and QTcP of -2.02 milliseconds (90%CI, -4.15 to 0.0679) were estimated, indicating there was no effect of TAK-931 on the QT intervals at a recommended phase 2 dose of 50 mg once daily for 14 days in a 21-day cycle.

Inhibition of the PLK1-Coupled Cell Cycle Machinery Overcomes Resistance to Oxaliplatin in Colorectal Cancer.

Dysregulation of the cell cycle machinery leads to genomic instability and is a hallmark of cancer associated with chemoresistance and poor prognosis in colorectal cancer (CRC). Identifying and targeting aberrant cell cycle machinery is expected to improve current therapies for CRC patients. Here,upregulated polo-like kinase 1 (PLK1) signaling, accompanied by deregulation of cell cycle-related pathways in CRC is identified. It is shown that aberrant PLK1 signaling correlates with recurrence and poor prognosis in CRC patients. Genetic and pharmacological blockade of PLK1 significantly increases the sensitivity to oxaliplatin in vitro and in vivo. Mechanistically, transcriptomic profiling analysis reveals that cell cycle-related pathways are activated by oxaliplatin treatment but suppressed by a PLK1 inhibitor. Cell division cycle 7 (CDC7) is further identified as a critical downstream effector of PLK1 signaling, which is transactivated via the PLK1-MYC axis. Increased CDC7 expression is also found to be positively correlated with aberrant PLK1 signaling in CRC and is associated with poor prognosis. Moreover, a CDC7 inhibitor synergistically enhances the anti-tumor effect of oxaliplatin in CRC models, demonstrating the potential utility of targeting the PLK1-MYC-CDC7 axis in the treatment of oxaliplatin-based chemotherapy.

Targeting CDC7 potentiates ATR-CHK1 signaling inhibition through induction of DNA replication stress in liver cancer.

BACKGROUND: Liver cancer is one of the most commonly diagnosed cancers and the fourth leading cause of cancer-related death worldwide. Broad-spectrum kinase inhibitors like sorafenib and lenvatinib provide only modest survival benefit to patients with hepatocellular carcinoma (HCC). This study aims to identify novel therapeutic strategies for HCC patients. METHODS: Integrated bioinformatics analyses and a non-biased CRISPR loss of function genetic screen were performed to identify potential therapeutic targets for HCC cells. Whole-transcriptome sequencing (RNA-Seq) and time-lapse live imaging were performed to explore the mechanisms of the synergy between CDC7 inhibition and ATR or CHK1 inhibitors in HCC cells. Multiple in vitro and in vivo assays were used to validate the synergistic effects. RESULTS: Through integrated bioinformatics analyses using the Cancer Dependency Map and the TCGA database, we identified ATR-CHK1 signaling as a therapeutic target for liver cancer. Pharmacological inhibition of ATR or CHK1 leads to robust proliferation inhibition in liver cancer cells having a high basal level of replication stress. For liver cancer cells that are resistant to ATR or CHK1 inhibition, treatment with CDC7 inhibitors induces strong DNA replication stress and consequently such drugs show striking synergy with ATR or CHK1 inhibitors. The synergy between ATR-CHK1 inhibition and CDC7 inhibition probably derives from abnormalities in mitosis inducing mitotic catastrophe. CONCLUSIONS: Our data highlights the potential of targeting ATR-CHK1 signaling, either alone or in combination with CDC7 inhibition, for the treatment of liver cancer.

MicroRNA-29b targeting of cell division cycle 7-related protein kinase (CDC7) regulated vascular smooth muscle cell (VSMC) proliferation and migration.

BACKGROUND: Proliferation and migration of vascular smooth muscle cells (VSMCs) are vital processes in vascular remodeling and pathology. This study aimed to explore the expression of miR-29b and cell division cycle 7-related protein kinase (CDC7) in patients with cerebral aneurysm (CA) and their effects on the proliferation and mobility of human umbilical artery smooth muscle cells (HUASMCs). METHODS: RNA levels of miR-29b and CDC7 were evaluated in the CA tissues and adjacent normal cerebral arteries from 18 patients undergoing surgery for CA rupture. The targeting of CDC7 by miR-29b was verified with luciferase reporter assay. Both CDC7 and miR-29b overexpression and silencing vectors were introduced to validate their effects on the proliferation and mobility of HUASMCs. RESULTS: The mRNA level of miR-29b was down-regulated (P<0.05), while the mRNA level of CDC7 was markedly elevated in CA patients (P<0.05). A Luciferase reporter assay showed CDC7 is a target gene of miR-29b, and miR-29b mimic down-regulated the mRNA and protein levels of CDC7 (P<0.05). Furthermore, miR-29b mimic inhibited, while miR-29b inhibitor or CDC7 over-expression promoted the proliferation and mobility of HUASMCs (P<0.05). CONCLUSIONS: miR-29-3p inhibits cell proliferation and mobility via directly targeting CDC7, which could be a potential therapeutic target for vascular dysfunction related diseases, including atherosclerosis and CA.

Cell division cycle 7 is a potential therapeutic target in oral squamous cell carcinoma and is regulated by E2F1.

Cell division cycle 7 (Cdc7) plays important roles in the regulation of the initiation of DNA replication throughout S phase. Whether inhibition of Cdc7 has a direct antitumour effect in oral squamous cell carcinoma (OSCC) remains unclear. In this study, XL413, a novel Cdc7 inhibitor, markedly inhibited the viability of OSCC cells but not that of non-tumour primary cells. There was a synergistic effect between XL413 and DNA-damaging agents (e.g. cisplatin and 5-fluorouracil) on OSCC in vitro and in vivo. Moreover, XL413 exhibited a notable antitumour effect on OSCC patients with high Cdc7 expression in mini patient-derived xenografts model. The proliferation was significantly inhibited in OSCC cells after Cdc7 silencing. Cdc7 knockdown significantly induced apoptosis in OSCC cell lines. Furthermore, we demonstrated that Cdc7 was overexpressed and transcriptionally regulated by E2F1 in OSCC by using chromatin immunoprecipitation and luciferase assays. Our results reveal that XL413 has an excellent antitumour effect in OSCC. Importantly, it does not inhibit the proliferation of non-tumour cells. These findings suggest that the overexpression of Cdc7 promotes progression in OSCC and that inhibition of Cdc7 is a very promising therapy for OSCC patients.

Mechanisms simultaneously regulate smooth muscle proliferation and differentiation.

Vascular smooth muscle cell (VSMC) differentiation and proliferation are two important physiological processes during vascular development. The phenotypic alteration from differentiated to proliferative VSMC contributes to the development of several major cardiovascular diseases including atherosclerosis, hypertension, restenosis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. Since the VSMC phenotype in these pathological conditions resembles that of developing VSMC during embryonic development, understanding of the molecular mechanisms that control VSMC differentiation will provide fundamental insights into the pathological processes of these cardiovascular diseases. Although VSMC differentiation is usually accompanied by an irreversible cell cycle exit, VSMC proliferation and differentiation occur concurrently during embryonic development. The molecular mechanisms simultaneously regulating these two processes, however, remain largely unknown. Our recent study demonstrates that cell division cycle 7, a key regulator of cell cycle, promotes both VSMC differentiation and proliferation through different mechanisms during the initial phase of VSMC differentiation. Conversely, Krüppel-like factor 4 appears to be a repressor for both VSMC differentiation and proliferation. This review attempts to highlight the novel role of cell division cycle 7 in TGF-ß-induced VSMC differentiation and proliferation. The role of Krüppel-like factor 4 in suppressing these two processes will also be discussed.

Assessing gene network stability and individual variability in the fathead minnow (Pimephales promelas) transcriptome.

Transcriptomics is increasingly used to assess biological responses to environmental stimuli and stressors such as aquatic pollutants. However, fundamental studies characterizing individual variability in mRNA levels are lacking, which currently limits the use of transcriptomics in environmental monitoring assessments. To address individual variability in transcript abundance, we performed a meta-analysis on 231 microarrays that were conducted in the fathead minnow (FHM), a widely used toxicological model. The mean variability for gene probes was ranked from most to least variable based upon the coefficient of variation. Transcripts that were the most variable in individual tissues included NADH dehydrogenase flavoprotein 1, GTPase IMAP family member 7-like and v-set domain-containing T-cell activation inhibitor 1-like while genes encoding ribosomal proteins (rpl24 and rpl36), basic transcription factor 3, and nascent polypeptide-associated complex alpha subunit were the least variable in individuals across a range of microarray experiments. Gene networks that showed high variability (based upon the variation in expression of individual members within the network) included cell proliferation, metabolism (steroid, lipids, and glucose), cell adhesion, vascularization, and regeneration while those that showed low variability (more stability) included mRNA and rRNA processing, regulation of translational fidelity, RNA splicing, and ribosome biogenesis. Real-time PCR was conducted on a subset of genes for comparison of variability collected from the microarrays. There was a significant positive relationship between the two methods when measuring individual variability, suggesting that variability detected in microarray data can be used to guide decisions on sample sizes for measuring transcripts in real-time PCR experiments. A power analysis revealed that measuring estrogen receptor ba (esrba) requires fewer biological replicates than that of estrogen receptor bb (esrbb) in the gonad and samples sizes required to detect a 50% change for reproductive-related transcripts is between 12 and 20. Characterizing individual variability at the molecular level will prove necessary as efforts are made toward integrating molecular tools into environmental risk assessments.