ATRX loss in glioma results in dysregulation of cell-cycle phase transition and ATM inhibitor radio-sensitization.

ATRX, a chromatin remodeler protein, is recurrently mutated in H3F3A-mutant pediatric glioblastoma (GBM) and isocitrate dehydrogenase (IDH)-mutant grade 2/3 adult glioma. Previous work has shown that ATRX-deficient GBM cells show enhanced sensitivity to irradiation, but the etiology remains unclear. We find that ATRX binds the regulatory elements of cell-cycle phase transition genes in GBM cells, and there is a marked reduction in Checkpoint Kinase 1 (CHEK1) expression with ATRX loss, leading to the early release of G2/M entry after irradiation. ATRX-deficient cells exhibit enhanced activation of master cell-cycle regulator ATM with irradiation. Addition of the ATM inhibitor AZD0156 doubles median survival in mice intracranially implanted with ATRX-deficient GBM cells, which is not seen in ATRX-wild-type controls. This study demonstrates that ATRX-deficient high-grade gliomas (HGGs) display Chk1-mediated dysregulation of cell-cycle phase transitions, which opens a window for therapies targeting this phenotype.

Chk1 and the Host Cell DNA Damage Response as a Potential Antiviral Target in BK Polyomavirus Infection.

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

Chk1 suppression leads to a reduction in the enhanced radiation-induced invasive capability on breast cancer cells.

Radiation therapy is generally effective for treating breast cancers. However, approximately 30% of patients with breast cancer experience occasional post-treatment local and distant metastasis. Low-dose (0.5 Gy) irradiation is a risk factor that promotes the invasiveness of breast cancers. Although an inhibitor of checkpoint kinase 1 (Chk1) suppresses the growth and motility of breast cancer cell lines, no study has investigated the effects of the combined use of a Chk1 inhibitor and radiation on cancer metastasis. Here, we addressed this question by treating the human breast cancer cell line MDA-MB-231 (in vitro) and mouse mammary tumor cell line 4 T1 (in vitro and in vivo) with γ-irradiation and the Chk1 inhibitor PD407824. Low-dose γ-irradiation promoted invasiveness, which was suppressed by PD407824. Comprehensive gene expression analysis revealed that low-dose γ-irradiation upregulated the mRNA and protein levels of S100A4, the both of which were downregulated by PD407824. We conclude that PD407824 suppresses the expression of S100A4. As the result, γ-irradiation-induced cell invasiveness were inhibited.

Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells.

Chk1 kinase is downstream of the ATR kinase in the sensing of improper replication. Previous cell culture studies have demonstrated that Chk1 is essential for replication. Indeed, Chk1 inhibitors are efficacious against tumors with high-level replication stress such as Myc-induced lymphoma cells. Treatment with Chk1 inhibitors also combines well with certain chemotherapeutic drugs, and effects associate with the induction of DNA damage and reduction of Chk1 protein levels. Most studies of Chk1 function have relied on the use of inhibitors. Whether or not a mouse or cancer cells could survive if a kinase-dead form of Chk1 is expressed has not been investigated before. Here, we generate a mouse model that expresses a kinase-dead (D130A) allele in the mouse germ line. We find that this mouse is overtly normal and does not have problems with erythropoiesis with aging as previously been shown for a mouse expressing one null allele. However, similar to a null allele, homozygous kinase-dead mice cannot be generated, and timed pregnancies of heterozygous mice suggest lethality of homozygous blastocysts at around the time of implantation. By breeding the kinase-dead Chk1 mouse with a conditional allele, we are able to demonstrate that expression of only one kinase-dead allele, but no wild-type allele, of Chek1 is lethal for Myc-induced cancer cells. Finally, treatment of melanoma cells with tumor-infiltrating T cells or CAR-T cells is effective even if Chk1 is inhibited, suggesting that Chk1 inhibitors can be safely administered in patients where immunotherapy is an essential component of the arsenal against cancer.

SPRTN protease and checkpoint kinase 1 cross-activation loop safeguards DNA replication.

The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease exhibit DPC-induced replication stress and genome instability, manifesting as premature ageing and liver cancer. Here, we provide a body of evidence suggesting that SPRTN activates the ATR-CHK1 phosphorylation signalling cascade during physiological DNA replication by proteolysis-dependent eviction of CHK1 from replicative chromatin. During this process, SPRTN proteolyses the C-terminal/inhibitory part of CHK1, liberating N-terminal CHK1 kinase active fragments. Simultaneously, CHK1 full length and its N-terminal fragments phosphorylate SPRTN at the C-terminal regulatory domain, which stimulates SPRTN recruitment to chromatin to promote unperturbed DNA replication fork progression and DPC repair. Our data suggest that a SPRTN-CHK1 cross-activation loop plays a part in DNA replication and protection from DNA replication stress. Finally, our results with purified components of this pathway further support the proposed model of a SPRTN-CHK1 cross-activation loop.

Inhibition of CHK 1 (Checkpoint Kinase 1) Elicits Therapeutic Effects in Pulmonary Arterial Hypertension.

OBJECTIVE: Pulmonary arterial hypertension (PAH) is a debilitating disease associated with progressive vascular remodeling of distal pulmonary arteries leading to elevation of pulmonary artery pressure, right ventricular hypertrophy, and death. Although presenting high levels of DNA damage that normally jeopardize their viability, pulmonary artery smooth muscle cells (PASMCs) from patients with PAH exhibit a cancer-like proproliferative and apoptosis-resistant phenotype accounting for vascular lumen obliteration. In cancer cells, overexpression of the serine/threonine-protein kinase CHK1 (checkpoint kinase 1) is exploited to counteract the excess of DNA damage insults they are exposed to. This study aimed to determine whether PAH-PASMCs have developed an orchestrated response mediated by CHK1 to overcome DNA damage, allowing cell survival and proliferation. Approach and Results: We demonstrated that CHK1 expression is markedly increased in isolated PASMCs and distal PAs from patients with PAH compared with controls, as well as in multiple complementary animal models recapitulating the disease, including monocrotaline rats and the simian immunodeficiency virus-infected macaques. Using a pharmacological and molecular loss of function approach, we showed that CHK1 promotes PAH-PASMCs proliferation and resistance to apoptosis. In addition, we found that inhibition of CHK1 induces downregulation of the DNA repair protein RAD 51 and severe DNA damage. In vivo, we provided evidence that pharmacological inhibition of CHK1 significantly reduces vascular remodeling and improves hemodynamic parameters in 2 experimental rat models of PAH. CONCLUSIONS: Our results show that CHK1 exerts a proproliferative function in PAH-PASMCs by mitigating DNA damage and suggest that CHK1 inhibition may, therefore, represent an attractive therapeutic option for patients with PAH.

ATR-mediated proteome remodeling is a major determinant of homologous recombination capacity in cancer cells.

The ATR kinase is crucial for genome maintenance, but the mechanisms by which ATR controls the DNA repair machinery are not fully understood. Here, we find that long-term chronic inhibition of ATR signaling severely impairs the ability of cells to utilize homologous recombination (HR)-mediated DNA repair. Proteomic analysis shows that chronic ATR inhibition depletes the abundance of key HR factors, suggesting that spontaneous ATR signaling enhances the capacity of cells to use HR-mediated repair by controlling the abundance of the HR machinery. Notably, ATR controls the abundance of HR factors largely via CHK1-dependent transcription, and can also promote stabilization of specific HR proteins. Cancer cells exhibit a strong dependency on ATR signaling for maintaining elevated levels of HR factors, and we propose that increased constitutive ATR signaling caused by augmented replication stress in cancer cells drives the enhanced HR capacity observed in certain tumor types. Overall, these findings define a major pro-HR function for ATR and have important implications for therapy by providing rationale for sensitizing HR-proficient cancer cells to PARP inhibitors.

Radiation-induced G2/M arrest rarely occurred in glioblastoma stem-like cells.

PURPOSE: The purpose of this study is to systematically study the cell-cycle alterations of glioblastoma stem-like cells (GSLCs) after irradiation, possibly enriching the mechanisms of radioresistance of GSLCs. MATERIALS AND METHODS: GSLCs were enriched and identified, and then the radioresistance of GSLCs was validated by analyzing cell survival, cell proliferation, and radiation-induced apoptosis. The discrepancy of the cell-cycle distribution and expression of cell-cycle-related proteins between GSLCs and glioblastoma differentiated cells (GDCs) after irradiation was completely analyzed. RESULTS: The survival fractions and the cell viabilities of GSLCs were significantly higher than those of GDCs after irradiation. Radiation-induced apoptosis was less prominent in GSLCs than in GDCs. After irradiation with high-dose X-rays, the percentages of GDCs in G2/M phase was evidently increased. However, radiation-induced G2/M arrest occurred less frequently in GSLCs, but S-phase arrest occurred in GSLCs after irradiation with 8 Gy. Further mechanistic studies showed that the expressions levels of Cdc25c, Cdc2, and CyclinB1 in GSLCs were not apparently changed after irradiation, while those of p-ATM and p-Chk1 were sharply increased after irradiation in GSLCs. The basal level of Cdc25c expression in GSLCs was much higher than that in GDCs. CONCLUSIONS: We explored the cell-cycle alterations and cell-cycle-related proteins expression levels in GSLCs after irradiation, providing a novel mechanism of radioresistance of GSLCs.

Reduced Expression of Mismatch Repair Genes MSH6/MSH2 Directly Promotes Pituitary Tumor Growth via the ATR-Chk1 Pathway.

Context: The mechanisms of pituitary adenoma (PA) pathogenesis and proliferation remain largely unknown. Objectives: To clarify the role of mismatch repair (MMR) genes in the molecular mechanism of PA proliferation. Design: We performed quantitative analyses by real-time polymerase chain reaction and immunohistochemistry to detect MMR gene and protein expression in human PAs (n = 47). We also performed correlation analyses of expression levels and tumor volume doubling time (TVDT; n = 31). Specifically, correlation analyses were performed between genes with significant correlation and ataxiatelangiectasia and Rad3-related (ATR) expression in cell-cycle regulatory mechanism ATR-checkpoint kinase 1 (Chk1) pathway (n = 93). We investigated the effect of reduced gene expression on cell proliferation and ATR gene expression in AtT-20ins cells and primary cultures of human PAs. Results: Expression of mutS homologs 6 and 2 (MSH6 and MSH2) was positively associated with TVDT (R = 0.52, P = 0.003, and R = 0.44, P = 0.01), as were the corresponding protein levels. Gene expression was positively associated with ATR expression (R = 0.47, P < 0.00001, and R = 0.49, P < 0.00001). In AtT-20ins, the reduction of MSH6 and/or MSH2 expression by small interfering RNA significantly promoted cell proliferation by decreasing ATR expression. This effect was also observed in primary culture. Conclusion: Reduction of MSH6 and MSH2 expression at the messenger RNA and protein levels could be involved in direct PA proliferation by promoting cell-cycle progression or decreasing the rate of apoptosis through interference with the function of the ATR-Chk1 pathway.

ATR/CHK1 inhibitors and cancer therapy.

The cell cycle checkpoint proteins ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its major downstream effector checkpoint kinase 1 (CHK1) prevent the entry of cells with damaged or incompletely replicated DNA into mitosis when the cells are challenged by DNA damaging agents, such as radiation therapy (RT) or chemotherapeutic drugs, that are the major modalities to treat cancer. This regulation is particularly evident in cells with a defective G1 checkpoint, a common feature of cancer cells, due to p53 mutations. In addition, ATR and/or CHK1 suppress replication stress (RS) by inhibiting excess origin firing, particularly in cells with activated oncogenes. Those functions of ATR/CHK1 make them ideal therapeutic targets. ATR/CHK1 inhibitors have been developed and are currently used either as single agents or paired with radiotherapy or a variety of genotoxic chemotherapies in preclinical and clinical studies. Here, we review the status of the development of ATR and CHK1 inhibitors. We also discuss the potential mechanisms by which ATR and CHK1 inhibition induces cell killing in the presence or absence of exogenous DNA damaging agents, such as RT and chemotherapeutic agents. Lastly, we discuss synthetic lethality interactions between the inhibition of ATR/CHK1 and defects in other DNA damage response (DDR) pathways/genes.

Checkpoint kinase 1 is essential for normal B cell development and lymphomagenesis.

Checkpoint kinase 1 (CHK1) is critical for intrinsic cell cycle control and coordination of cell cycle progression in response to DNA damage. Despite its essential function, CHK1 has been identified as a target to kill cancer cells and studies using Chk1 haploinsufficient mice initially suggested a role as tumor suppressor. Here, we report on the key role of CHK1 in normal B-cell development, lymphomagenesis and cell survival. Chemical CHK1 inhibition induces BCL2-regulated apoptosis in primary as well as malignant B-cells and CHK1 expression levels control the timing of lymphomagenesis in mice. Moreover, total ablation of Chk1 in B-cells arrests their development at the pro-B cell stage, a block that, surprisingly, cannot be overcome by inhibition of mitochondrial apoptosis, as cell cycle arrest is initiated as an alternative fate to limit the spread of damaged DNA. Our findings define CHK1 as essential in B-cell development and potent target to treat blood cancer.

Alterations in protein phosphorylation in the amygdala of the 5XFamilial Alzheimer's disease animal model.

Alzheimer's disease is the most common disease underlying dementia in humans. Two major neuropathological hallmarks of AD are neuritic plaques primarily composed of amyloid beta peptide and neurofibrillary tangles primarily composed of hyperphosphorylated tau. In addition to impaired memory function, AD patients often display neuropsychiatric symptoms and abnormal emotional states such as confusion, delusion, manic/depressive episodes and altered fear status. Brains from AD patients show atrophy of the amygdala which is involved in fear expression and emotional processing as well as hippocampal atrophy. However, which molecular changes are responsible for the altered emotional states observed in AD remains to be elucidated. Here, we observed that the fear response as assessed by evaluating fear memory via a cued fear conditioning test was impaired in 5XFamilial AD (5XFAD) mice, an animal model of AD. Compared to wild-type mice, 5XFAD mice showed changes in the phosphorylation of twelve proteins in the amygdala. Thus, our study provides twelve potential protein targets in the amygdala that may be responsible for the impairment in fear memory in AD.

Mus81 knockdown sensitizes colon cancer cells to chemotherapeutic drugs by activating CHK1 pathway.

PURPOSE: The inhibition of Mus81, a critical DNA repair gene, is recently related to the chemosensitivity of several human cancer cells such as hepatocellular carcinoma (HCC) cells. However, the role of Mus81 knockdown in chemotherapy response of colon cancer cells remains largely unknown. METHODS AND MATERIALS: The effects of Mus81 knockdown by lentivirus-mediated short hairpin RNA in sensitivity of HCT116 and LS180 colon cancer cell lines to four therapeutic drugs, including cisplatin (CDDP), were evaluated by MTT assay as well as a mouse model. Apoptosis and cell cycle distribution of HCT116 cell line was detected by flow cytometric analysis. Western blot was also employed to determine the expression of CHK1 pathway and apoptosis-related proteins in HCT116 cells and the xenograft mouse tumors. RESULTS: Mus81 knockdown could significantly improve the chemosensitivity of colon cancer cells in vitro and in vivo, especially to CDDP. Mus81 knockdown also induced S phase arrest and elevated apoptosis in CDDP treated HCT116 cells through activating CHK1/CDC25A/CDK2 and CHK1/p53/Bax pathways, while these effects could be counteracted by CHK1 inhibition. CONCLUSION: Mus81 knockdown improves the chemosensitivity of colon cancer cells by inducing S phase arrest and promoting apoptosis through activating CHK1 pathway.

Cell cycle proteins as promising targets in cancer therapy.

Cancer is characterized by uncontrolled tumour cell proliferation resulting from aberrant activity of various cell cycle proteins. Therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrate that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. By contrast, many cancers are uniquely dependent on these proteins and hence are selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. In this Review, we focus on proteins that directly regulate cell cycle progression (such as cyclin-dependent kinases (CDKs)), as well as checkpoint kinases, Aurora kinases and Polo-like kinases (PLKs). We discuss the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as the future therapeutic potential of various cell cycle inhibitors.

Defective DNA repair increases susceptibility to senescence through extension of Chk1-mediated G2 checkpoint activation.

Susceptibility to senescence caused by defective DNA repair is a major hallmark of progeroid syndrome patients, but molecular mechanisms of how defective DNA repair predisposes to senescence are largely unknown. We demonstrate here that suppression of DNA repair pathways extends the duration of Chk1-dependent G2 checkpoint activation and sensitizes cells to senescence through enhancement of mitosis skipping. Extension of G2 checkpoint activation by introduction of the TopBP1 activation domain and the nondegradable mutant of Claspin sensitizes cells to senescence. In contrast, a shortening of G2 checkpoint activation by expression of SIRT6 or depletion of OTUB2 reduces susceptibility to senescence. Fibroblasts from progeroid syndromes tested shows a correlation between an extension of G2 checkpoint activation and an increase in the susceptibility to senescence. These results suggest that extension of G2 checkpoint activation caused by defective DNA repair is critical for senescence predisposition in progeroid syndrome patients.