Role Of Chk1 Research Articles

Functional analysis of chromatin-associated proteins in Sordaria macrospora reveals similar roles for RTT109 and ASF1 in development and DNA damage response.

We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA damage resistance in the ascomycete Sordaria macrospora. ASF1 is known to be involved in nucleosome assembly and disassembly, binding histones H3 and H4 during transcription, replication and DNA repair and has direct and indirect roles in histone recycling and modification as well as DNA methylation, acting as a chromatin modifier hub for a large network of chromatin-associated proteins. Here, we functionally characterized two of these proteins, RTT109 and CHK2. RTT109 is a fungal-specific histone acetyltransferase, while CHK2 is an ortholog to PRD-4, a checkpoint kinase of Neurospora crassa that performs similar cell cycle checkpoint functions as yeast RAD53. Through the generation and characterization of deletion mutants, we discovered striking similarities between RTT109 and ASF1 in terms of their contributions to sexual development, histone acetylation, and protection against DNA damage. Phenotypic observations revealed a developmental arrest at the same stage in Δrtt109 and Δasf1 strains, accompanied by a loss of H3K56 acetylation, as detected by western blot analysis. Deletion mutants of rtt109 and asf1 are sensitive to the DNA damaging agent methyl methanesulfonate, but not hydroxyurea. In contrast, chk2 mutants are fertile and resistant to methyl methanesulfonate, but not hydroxyurea. Our findings suggest a close functional association between ASF1 and RTT109 in the context of development, histone modification, and DNA damage response, while indicating a role for CHK2 in separate pathways of the DNA damage response.

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.

Immunohistochemistry analysis of checkpoint kinase 2 in oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) is the most frequently occurring malignant tumor of the head and neck region. Chk2 (Checkpoint kinase 2) is considered a tumor suppressor gene that acts on the cellular response to DNA damage. However, the role of Chk2 in OSCC prognosis is not yet fully understood. The objective of this study was to evaluate Chk2 immunoexpression in OSCC and to elucidate the association between its expression and clinicopathological parameters of prognostic importance, including overall survival, disease-free survival, and metastasis-free survival.MethodsChk2 expression was analyzed in 101 samples from patients with OSCC using immunohistochemistry. We stratified the patients into high expression (> 66% of cells positive for Chk2) and low expression (< 66%) groups.ResultsChk2 showed high expression in 57.43% of OSCC. In our study, the expression of Chk2 did not correlate with any of the prognostic parameters evaluated. There was no difference between overall survival, metastasis-free survival, and disease-free survival according to Chk2 expression.ConclusionDespite the great importance of Chk2 in the development of different types of cancer, our findings do not favor Chk2 as a prognostic marker in oral squamous cell carcinoma.

Glucocorticoids and checkpoint tyrosine kinase inhibitors stimulate rat pancreatic beta cell proliferation differentially.

Cell therapy for diabetes could benefit from the identification of small-molecule compounds that increase the number of functional pancreatic beta cells. Using a newly developed screening assay, we previously identified glucocorticoids as potent stimulators of human and rat beta cell proliferation. We now compare the stimulatory action of these steroid hormones to a selection of checkpoint tyrosine kinase inhibitors that were also found to activate the cell cycle-in beta cells and analyzed their respective effects on DNA-synthesis, beta cell numbers and expression of cell cycle regulators. Our data using glucocorticoids in combination with a receptor antagonist, mifepristone, show that 48h exposure is sufficient to allow beta cells to pass the cell cycle restriction point and to become committed to cell division regardless of sustained glucocorticoid-signaling. To reach the end-point of mitosis another 40h is required. Within 14 days glucocorticoids stimulate up to 75% of the cells to undergo mitosis, which indicates that these steroid hormones act as proliferation competence-inducing factors. In contrast, by correlating thymidine-analogue incorporation to changes in absolute cell numbers, we show that the checkpoint kinase inhibitors, as compared to glucocorticoids, stimulate DNA-synthesis only during a short time-window in a minority of cells, insufficient to give a measurable increase of beta cell numbers. Glucocorticoids, but not the kinase inhibitors, were also found to induce changes in the expression of checkpoint regulators. Our data, using checkpoint kinase-specific inhibitors further point to a role for Chk1 and Cdk1 in G1/S transition and progression of beta cells through the cell cycle upon stimulation with glucocorticoids.

Replication stress response in cancer stem cells as a target for chemotherapy.

Cancer stem cells (CSCs) are subpopulations of multipotent stem cells (SCs) responsible for the initiation, long-term clonal maintenance, growth and spreading of most human neoplasms. Reportedly, CSCs share a very robust DNA damage response (DDR) with embryonic and adult SCs, which allows them to survive endogenous and exogenous genotoxins. A range of experimental evidence indicates that CSCs have high but heterogeneous levels of replication stress (RS), arising from, and being boosted by, endogenous causes, such as specific genetic backgrounds (e.g., p53 deficiency) and/or aberrant karyotypes (e.g., supernumerary chromosomes). A multipronged RS response (RSR) is put in place by CSCs to limit and ensure tolerability to RS. The characteristics of such dedicated cascade have two opposite consequences, both relevant for cancer therapy. On the one hand, RSR efficiency often increases the reliance of CSCs on specific DDR components. On the other hand, the functional redundancy of pathways of the RSR can paradoxically promote the acquisition of resistance to RS- and/or DNA damage-inducing agents. Here, we provide an overview of the molecular mechanisms of the RSR in cancer cells and CSCs, focusing on the role of CHK1 and some emerging players, such as PARP1 and components of the homologous recombination repair, whose targeting can represent a long-term effective anti-CSC strategy.

RA-Induced Transcriptional Silencing of Checkpoint Kinase-2 through Promoter Methylation by Dnmt3b Is Required for Neuronal Differentiation of P19 Cells

In a previous study, we identified several novel targets of Dnmt3b using a chromatin library from retinoic acid (RA)-treated P19 cells. The present study describes the regulation of expression and function of checkpoint kinase (Chk2), which was one of the target genes of Dnmt3b. Chromatin immunoprecipitation followed by quantitative PCR analysis showed that recruitment of Dnmt3b on Chk2 promoter is induced following RA treatment of P19 cells. Both bisulfite genomic sequence and COBRA analyses showed that the methylation level of Chk2 promoter is progressively increased during RA-induced neuronal differentiation of P19 cells. Concomitantly, both mRNA and protein expression of Chk2 are reduced as determined by real-time PCR and Western blot analysis, respectively. Suppression of Dnmt3b expression by lentiviral-mediated shRNA resulted in increased expression and reduced methylation of Chk2, which clearly showed that Dnmt3b is responsible for transcriptional silencing of Chk2 gene in RA-treated P19 cells. Neuronal differentiation of P19 cells was inhibited upon enforced Chk2 expression in P19 cells, which showed that the decrease in endogenous expression of Chk2 is essential for normal differentiation. Ectopic Chk2 expression also negatively regulated cell cycle arrest and apoptosis following RA treatment, which could also contribute to impaired neuronal differentiation. Together, this study described the regulation of Chk2 expression through promoter methylation and also presented a novel role of Chk2 during neuronal differentiation, which is independent of its previously known function in DNA damage response.

Abstract IA13: CHK1 inhibitors: From concept to clinic

Abstract Each cell in the human body is subjected daily to both exogenous and endogenous insults that result in DNA damage. This assault on the human genome can lead to the formation of many different types of DNA lesions such as single strand breaks (SSBs), double strand breaks (DSBs), base modifications and base mismatches that are repaired by a number of distinct but interrelated mechanisms. However, the failure to detect and repair these lesions can lead to cells with intrinsically high levels of DNA damage, gene mutations and often even larger genomic changes including chromosomal aberrations. These changes within the DNA may all contribute to the genome becoming unstable, such that there is an elevated frequency of genomic alterations. This genomic instability has now been acknowledged as a hallmark of cancer. Detection and response to DNA Damage is therefore essential for cells and is undertaken by the DNA damage response (DDR) pathway, an intracellular signaling network that coordinates and regulates the cellular activities that can detect and repair damaged DNA. A key component of this is pathway is the serine/threonine kinase CHK1. This kinase has the ability, through substrate phosphorylation to halt the cell division cycle, in particular at S and G2/M checkpoints. This gives the cell time to either repair the damaged DNA and re-initiate the cell cycle or if effective DNA repair is not possible, to induce cell senescence or cell death. The DDR is important for both normal and cancer cells. Cancer cells however, often have gene alterations that cause defects in one or more cell cycle checkpoints or in DNA repair. An important gene in this context is TP53 which encodes the p53 protein that can activate the G1/S cell cycle checkpoint in response to DNA damage. A hallmark of many tumors is the lack of functional p53 protein with a consequent loss of the G1/S checkpoint leading to a potential increased reliance on the S and G2/M checkpoints for survival. It was therefore initially hypothesized that CHK1 inhibitors may selectively enhance the activity of genotoxic agents in tumors, in particular by abrogating the G2 checkpoint, while normal cells would be rescued due to their competent DDR. This led to development a number of CHK1 inhibitors that are progressing towards, or are currently undergoing, clinical evaluation in combination with specific genotoxic cancer drugs. These include the potent, selective and orally bioavailable CHK1 inhibitor CCT245737, which is now scheduled for Phase I clinical trial and will be discussed during this presentation. Research now suggests that the role of CHK1 during S-phase may also be important when combining CHK1 inhibitors with selected chemotherapeutic agents and this will also be discussed. A key role for CHK1 during S phase is upon replication stress, where it stabilizes and preserves replication fork complexes thus preventing catastrophic replication fork collapse, until replication can be re-initiated. As well as suffering from genome instability, many cancer cells exhibit high levels of replication stress, which is thought to be due to oncogene-driven proliferation. Therefore tumors with high levels of replication stress will be dependent on functional CHK1. This provides a potential monotherapy strategy for CHK1 inhibitors in selected adult and pediatric cancer settings that are oncogene-driven and/or exhibit replication stress, which will be discussed in this presentation. Citation Format: Michelle D. Garrett. CHK1 inhibitors: From concept to clinic. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA13.

Abstract 2729: Estrogen receptor-negative breast cancer cell lines exhibit hypersensitivity to the CHK1 inhibitor LY2606368

Abstract Checkpoint kinase 1 (CHK1) inhibitors are currently under investigation as chemopotentiating agents due to the role of CHK1 in establishing DNA damage checkpoints in the cell cycle. A novel CHK1/CHK2 inhibitor, LY2606368, as a single agent causes replication catastrophe, DNA double strand breaks and apoptosis (King C et al. Mol Cancer Ther. 2015). Accordingly, LY2606368 is currently in clinical development as a single agent and in combination with both cytotoxic and targeted agents. As subsets of breast cancers exhibit genomic instability and DNA repair deficiencies, we assessed the effect of LY2606368 as a single agent on breast cancer cell lines. We characterized the IC50's for growth inhibition by LY2606368 of a large panel of cell lines assembled on the basis of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression status. Triple negative breast cancer (TNBC) is a subtype of breast cancer that is pathologically negative for expression of ER/PR and HER2 protein. As previously reported for other CHK1 inhibitors (Bryant C et al. BMC Cancer. 2014; Albiges L et al. Breast. 2014), TNBC cell lines exhibited high sensitivity to LY2606368. Interestingly, hypersensitivity to LY2606368 was observed in ER-negative cells, regardless of HER2 status. In addition, ER-positive cells were comparatively resistant suggesting that high sensitivity to LY2606368 occurs in the absence of ER and is not restricted to TNBC. No correlation was found between TP53 mutational status and sensitivity to LY2606368. Consistent with the observed hypersensitivity, ER-negative cell lines exposed to LY2606368 exhibited high levels of γH2AX and phospho-Ser1981-ATM demonstrating appearance of DNA double strand breaks. The concomitant appearance of phospho-Ser345-Chk1 marked aborted checkpoint activation by upstream detector/effector kinases (e.g. ATR, ATM). ER-positive cells did not engage in initiation of the DNA damage response or significant checkpoint activation when exposed to comparable doses of LY2606368. Collectively, these results suggest that the CHK1 inhibitor LY2606368 is likely to be more cytotoxic in ER-negative breast cancer types. Homologous recombination and other DNA repair deficiencies can explain the need for CHK1-dependent checkpoint activation during each replication cycle. On the other hand, ER-positive breast cancers might be less sensitive to monotherapy because of the absence of unresolved DNA damaging events that create checkpoint dependency. In summary, triple negative status does not directly determine the sensitivity of breast cancer cell lines to the CHK1 inhibitor LY2606368, although TNBC cells are in general hypersensitive. The absence of ER might be a more promising marker of sensitivity to LY2606368 as a monotherapy in breast cancer. Citation Format: Jean-Bernard Lazaro, Kalindi Parmar, Geoffrey I. Shapiro, Alan D. D’Andrea. Estrogen receptor-negative breast cancer cell lines exhibit hypersensitivity to the CHK1 inhibitor LY2606368. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2729.

Abstract 2724: Collaborative regulation of cell cycle progression by Chk1 and E2F1 in small cell lung cancer cells

Abstract Introduction Small cell lung cancer (SCLC) harbors very frequent mutations in p53 and Rb, key cell cycle regulators in normal cells. In absence of such tumor suppressors, SCLC cells rely on Chk1 for cell cycle arrest in the event of DNA damage. High expression of E2F1 and Chk1 have been found in SCLCs, which is associated with poor prognosis in various solid tumors. In human SCLC cell lines, Chk1 inhibition sensitizes chemotherapy agents. The interaction between Chk1 and E2F1 is understudied. Here, we report a collaborative role of Chk1 and E2F1 in the cell cycle regulation in SCLC cells. Methods Three Rb-mutated human SCLC cell lines (GLC4, NCI-H128, and NCI-H209) were used. GLC4 carries mutant p53; H209 and H128 are wild type. Chk1 inhibition was achieved by either siRNA knockdown or LY2940930; E2F1 was knocked down by siRNA. Ectopic overexpression of Chk1 or E2F1 was achieved using Lonza electroporation kit. Cell viability was measured by Cell-Titer Glo assay. Cell cycle analysis was assayed by PI staining and FACS. Western blotting was used to evaluate caspase activation and other signaling proteins. Results Chk1 inhibition by siRNA knockdown or LY2940930 treatment enhanced cisplatin cytotoxicity in SCLC cell lines, regardless of p53 status. Also, a significant decrease of E2F1 protein was observed following Chk1 inhibition in these cells. Knockdown of E2F1 by siRNA enhanced the cytotoxic activity of cisplatin in the GLC4 and H209 cells. In GLC4 cells, Chk1 siRNA combined with cisplatin treatment caused activation of caspase-2 and caspase-3 as well as the cleavage of BID. Interestingly, knocking down caspase-2 reversed caspase-3 activation resulting from the combination of Chk1 siRNA and cisplatin, and restored the level of E2F1 protein. In contrast, ectopic overexpression of Chk1 resulted in an increase of E2F1, while increase of Chk1 expression was also noticeable upon E2F1 overexpression. It has been shown that Chk1 inhibition can abrogate cell cycle arrest and increase phospho-histone H3 expression (mitotic marker). We found that E2F1-overexpressing GLC4 and H128 cells displayed higher levels of phospho-histone H3 when treated with LY2940930, compared to that of Mock-transfected controls. Furthermore, exogenous E2F1 overexpression significantly reduced the IC50 of cisplatin (1331nM vs. 3698nM; p = 0.0028 by paired t-test) in GLC4 cells, but not that of LY2940930 (14nM vs. 19nM; p = 0.25). Conclusions Either downregulation of Chk1 or overexpression of E2F1 leads to cell cycle progression, resulting in more cell death when combined with DNA damaging agents. Decrease of E2F1 by Chk1 inhibition might represent a mechanism of self-protection to avoid excessive mitosis after DNA damage, and this regulation is likely involving caspase-2. Further exploration of Chk1 and E2F1 regulatory mechanisms is warranted in SCLC. Citation Format: Wei-Hsun Hsu, In-Gyu Kim, Guanhua Rao, Justine McCutcheon, Shuo-Tse Hsu, Yu-Wen Zhang, Giuseppe Giaccone. Collaborative regulation of cell cycle progression by Chk1 and E2F1 in small cell lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2724.

CHK1 regulates NF-κB signaling upon DNA damage in p53- deficient cells and associated tumor-derived microvesicles

The recently discovered CHK1-Suppressed (CS) pathway is activated by inhibition or loss of the checkpoint kinase CHK1, promoting an apoptotic response to DNA damage mediated by caspase-2 in p53-deficient cells. Although functions of the CS-pathway have been investigated biochemically, it remains unclear whether and how CHK1 inhibition can be regulated endogenously and whether this constitutes a key component of the DNA damage response (DDR). Here, we present data that define the first endogenous activation of the CS-pathway whereby, upon DNA damage, wild type p53 acts as an endogenous regulator of CHK1 levels that modulates caspase-2 activation. Moreover, we demonstrate that persistence of CHK1 levels in response to DNA damage in p53-deficient cancer cells, leads to CHK1-mediated activation of NF-κB and induction of NF-κB-regulated genes in cells and in associated tumor-derived microvesicles (TMVs), both of which are abrogated by loss or inhibition of CHK1. These data define a novel role for CHK1 in the DDR pathway as a regulator NF-κB activity. Our data provide evidence that targeting CHK1 in p53-deficient cancers may abrogate NF-κB signaling that is associated with increased cellular survival and chemoresistance.

Human papillomaviruses activate and recruit SMC1 cohesin proteins for the differentiation-dependent life cycle through association with CTCF insulators.

Human papillomaviruses infect stratified epithelia and link their productive life cycle to the differentiation state of the host cell. Productive viral replication or amplification is restricted to highly differentiated suprabasal cells and is dependent on the activation of the ATM DNA damage pathway. The ATM pathway has three arms that can act independently of one another. One arm is centered on p53, another on CHK2 and a third on SMC1/NBS1 proteins. A role for CHK2 in HPV genome amplification has been demonstrated but it was unclear what other factors provided important activities. The cohesin protein, SMC1, is necessary for sister chromatid association prior to mitosis. In addition the phosphorylated form of SMC1 plays a critical role together with NBS1 in the ATM DNA damage response. In normal cells, SMC1 becomes phosphorylated in response to radiation, however, in HPV positive cells our studies demonstrate that it is constitutively activated. Furthermore, pSMC1 is found localized in distinct nuclear foci in complexes with γ-H2AX, and CHK2 and bound to HPV DNA. Importantly, knockdown of SMC1 blocks differentiation-dependent genome amplification. pSMC1 forms complexes with the insulator transcription factor CTCF and our studies show that these factors bind to conserved sequence motifs in the L2 late region of HPV 31. Similar motifs are found in most HPV types. Knockdown of CTCF with shRNAs blocks genome amplification and mutation of the CTCF binding motifs in the L2 open reading frame inhibits stable maintenance of viral episomes in undifferentiated cells as well as amplification of genomes upon differentiation. These findings suggest a model in which SMC1 factors are constitutively activated in HPV positive cells and recruited to viral genomes through complex formation with CTCF to facilitate genome amplification. Our findings identify both SMC1 and CTCF as critical regulators of the differentiation-dependent life cycle of high-risk human papillomaviruses.

CHK1-driven histone H3.3 serine 31 phosphorylation is important for chromatin maintenance and cell survival in human ALT cancer cells.

Human ALT cancers show high mutation rates in ATRX and DAXX. Although it is well known that the absence of ATRX/DAXX disrupts H3.3 deposition at heterochromatin, its impact on H3.3 deposition and post-translational modification in the global genome remains unclear. Here, we explore the dynamics of phosphorylated H3.3 serine 31 (H3.3S31ph) in human ALT cancer cells. While H3.3S31ph is found only at pericentric satellite DNA repeats during mitosis in most somatic human cells, a high level of H3.3S31ph is detected on the entire chromosome in ALT cells, attributable to an elevated CHK1 activity in these cells. Drug inhibition of CHK1 activity during mitosis and expression of mutant H3.3S31A in these ALT cells result in a decrease in H3.3S31ph levels accompanied with increased levels of phosphorylated H2AX serine 139 on chromosome arms and at the telomeres. Furthermore, the inhibition of CHK1 activity in these cells also reduces cell viability. Our findings suggest a novel role of CHK1 as an H3.3S31 kinase, and that CHK1-mediated H3.3S31ph plays an important role in the maintenance of chromatin integrity and cell survival in ALT cancer cells.

CHK2 kinase in the DNA damage response and beyond.

The serine/threonine kinase CHK2 is a key component of the DNA damage response. In human cells, following genotoxic stress, CHK2 is activated and phosphorylates >20 proteins to induce the appropriate cellular response, which, depending on the extent of damage, the cell type, and other factors, could be cell cycle checkpoint activation, induction of apoptosis or senescence, DNA repair, or tolerance of the damage. Recently, CHK2 has also been found to have cellular functions independent of the presence of nuclear DNA lesions. In particular, CHK2 participates in several molecular processes involved in DNA structure modification and cell cycle progression. In this review, we discuss the activity of CHK2 in response to DNA damage and in the maintenance of the biological functions in unstressed cells. These activities are also considered in relation to a possible role of CHK2 in tumorigenesis and, as a consequence, as a target of cancer therapy.

Abstract 2766: Inhibition of Chk1 and Wee1 as a new therapeutic approach in Mantle Cell Lymphoma

Abstract Mantle Cell Lymphoma (MCL) is an aggressive lymphoma, for which there is not yet a standard therapy. Deregulation of cell cycle is its main pathogenic feature due to the presence of the t(11;14)(q13;q32) translocation, which leads to cyclin D1 over-expression and to frequent alterations in cell cycle and DNA damage response. Chk1 and Wee1 proteins regulate replication initiation, replication fork stability, and mitotic entry. Combined treatment of Chk1 and Wee1 inhibitors has a strong synergistic cytotoxic effect in solid tumors, but little is known on their effect in hematologic cancer such as lymphomas. The effects of a Chk1 inhibitor (PF-00477736) and a Wee1 inhibitor (MK-1775) alone or in combination was investigated in a panel of 10 MCL cell lines. The PF-00477736 median IC50 value was 30 nM, significantly lower than in 9 solid tumor models (337 nM, p=0.0041) and in 8 hematologic tumors (184 nM, p= 0.0057). The MK-1775 median IC50 value was 106 nM, significantly lower than in solid tumors (615 nM, p&amp;lt;0.0001), but not than in other hematologic cells (287 nM). A strong synergism of the combination was observed in all MCL cell lines. Detailed molecular analysis showed that the combination caused a general deregulation of cell cycle with an increased activity of CDK2 and CDK1 and an activation of apoptosis. Inhibition of the CDK4/6-CyclinD1 complex with the selective inhibitor PD0332991 either strongly or slightly neutralize the cytotoxic effect of PF-00477736 and MK-1775 respectively in Jeko-1 MCL cells. In addition, the sensitivity to PF-00477736 and MK-1775 was higher in 2 multiple myeloma (MM) cell lines bearing the t(11;14) deregulating cyclinD1 than in 3 MM cell lines without the translocation (with median IC50 values 40 times lower for PF-00477736 and two times lower for MK-1775). The combination of Chk1 and Wee1 inhibitors was then tested in vivo in nude mice bearing Jeko-1 MCL and important tumor regressions were observed at drug doses lower than the ones used in solid tumors xenografts (best T/C%=0.54%). Tumor samples obtained from xenografts untreated, treated with single drug or the combination were profiled for gene expression and the analysis corroborated that genes involved in apoptosis were up-regulated after combined treatment in vivo. In conclusion, our data provide a strong preclinical evidence for the role of Chk1 and Wee1 inhibitors as new therapeutic approach in MCL and warrant investigation in clinical setting. Citation Format: Laura Carrassa, Rosaria Chilà, Alessandra Basana, Francesca Ricci, Federica Guffanti, Monica Lupi, Andrea Rinaldi, Luciano Cascione, Francesco Bertoni, Massimo Broggini, Giovanna Damia. Inhibition of Chk1 and Wee1 as a new therapeutic approach in Mantle Cell Lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2766. doi:10.1158/1538-7445.AM2014-2766

DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B.

The disruption of DNA replication in cells triggers checkpoint responses that slow-down S-phase progression and protect replication fork integrity. These checkpoints are also determinants of cell fate and can help maintain cell viability or trigger cell death pathways. CHK1 has a pivotal role in such S-phase responses. It helps maintain fork integrity during replication stress and protects cells from several catastrophic fates including premature mitosis, premature chromosome condensation and apoptosis. Here we investigated the role of CHK1 in protecting cancer cells from premature mitosis and apoptosis. We show that premature mitosis (characterized by the induction of histone H3 phosphorylation, aberrant chromatin condensation, and persistent RPA foci in arrested S-phase cells) is induced in p53-deficient tumour cells depleted of CHK1 when DNA synthesis is disrupted. These events are accompanied by an activation of Aurora kinase B in S-phase cells that is essential for histone H3 Ser10 phosphorylation. Histone H3 phosphorylation precedes the induction of apoptosis in p53−/− tumour cell lines but does not appear to be required for this fate as an Aurora kinase inhibitor suppresses phosphorylation of both Aurora B and histone H3 but has little effect on cell death. In contrast, only a small fraction of p53+/+ tumour cells shows this premature mitotic response, although they undergo a more rapid and robust apoptotic response. Taken together, our results suggest a novel role for CHK1 in the control of Aurora B activation during DNA replication stress and support the idea that premature mitosis is a distinct cell fate triggered by the disruption of DNA replication when CHK1 function is suppressed.

Roles of Chk1 in cell biology and cancer therapy

The evolutionally conserved DNA damage response (DDR) and cell cycle checkpoints preserve genome integrity. Central to these genome surveillance pathways is a protein kinase, Chk1. DNA damage induces activation of Chk1, which then transduces the checkpoint signal and facilitates cell cycle arrest and DNA damage repair. Significant progress has been made recently toward our understanding of Chk1 regulation and its implications in cancer etiology and therapy. Specifically, a model that involves both spatiotemporal and conformational changes of proteins has been proposed for Chk1 activation. Further, emerging evidence suggests that Chk1 does not appear to be a tumor suppressor; instead, it promotes tumor growth and may contribute to anticancer therapy resistance. Recent data from our laboratory suggest that activating, but not inhibiting, Chk1 in the absence of chemotherapy might represent an innovative approach to suppress tumor growth. These findings suggest unique regulation of Chk1 in cell biology and cancer etiology, pointing to novel strategies for targeting Chk1 in cancer therapy.

The Co-Repressor SMRT Delays DNA Damage-Induced Caspase Activation by Repressing Pro-Apoptotic Genes and Modulating the Dynamics of Checkpoint Kinase 2 Activation

Checkpoint kinase 2 (Chk2) is a major regulator of DNA damage response and can induce alternative cellular responses: cell cycle arrest and DNA repair or programmed cell death. Here, we report the identification of a new role of Chk2 in transcriptional regulation that also contributes to modulating the balance between survival and apoptosis following DNA damage. We found that Chk2 interacts with members of the NCoR/SMRT transcriptional co-regulator complexes and serves as a functional component of the repressor complex, being required for recruitment of SMRT on the promoter of pro-apoptotic genes upon DNA damage. Thus, the co-repressor SMRT exerts a critical protective action against genotoxic stress-induced caspase activation, repressing a functionally important cohort of pro-apoptotic genes. Amongst them, SMRT is responsible for basal repression of Wip1, a phosphatase that de-phosphorylates and inactivates Chk2, thus affecting a feedback loop responsible for licensing the correct timing of Chk2 activation and the proper execution of the DNA repair process.

Abstract 619: CHK2 and DNAPKc deficiency reduces DNA repair capacity and increases transformation efficiency in human bronchial epithelial cells.

Abstract Reduced DNA repair capacity (DRC) and polymorphisms in genes involved in the repair pathways are associated with increased risk for lung cancer. Studies from our lab have demonstrated an association between increased double strand breaks (DSB) and increased risk for gene promoter hypermethylation. The goal of this study was to identify genes in the DSB repair pathway that may be rate limiting for DNA repair and to determine the effect of gene knockdown on genomic stability, DRC, gene promoter methylation, and cellular transformation. CHK2 is an important DNA damage sensor and cell cycle regulator gene that is important in maintaining genomic stability. DNAPKc is a kinase within the non-homologous end joining (NHEJ) pathway that participates in DNA repair by inducing conformational changes that allows access to DSB sites, and reduced activity is associated with lung cancer. Expression of CHK2 was reduced in 4 of 10 tumor-derived lung cancer cell lines, while 3 cell lines had undetectable levels of DNAPKc. We developed an in vitro model using human telomerase/cyclin dependent kinase 4-immortalized human bronchial epithelial cell lines (HBECs) to identify key molecular changes that drive transformation and clonal outgrowth of preneoplastic lung epithelial cells during exposure to carcinogens. CHK2 and DNAPKc were stably knocked down in HBEC2 and HBEC3 to determine their role in DRC, genomic instability and transformation. Knockdown of neither CHK2 nor DNAPKc affected microsatellite instability, nor was deficiency of either gene sufficient to induce spontaneous transformation in HBEC2 and HBEC3. Knockdown of CHK2 or DNAPKc decreased DRC in HBEC2 and HBEC3 in response to bleomycin, (a DSB-inducing carcinogen) indicating an important role for both genes in DSB repair. Knockdown cell lines were treated with bleomycin once a week for 12 weeks to determine the effect of gene deficiency on transformation. DNAPKc deficiency increased transformation efficiency in HBEC2 and HBEC3 by 14- and 4.5- fold, respectively, while CHK2 deficiency increased transformation efficiency in HBEC3 by 6- fold compared to bleomycin treated controls. Studies are ongoing to determine changes induced in the transcriptome and methylome in DNA repair-deficient cells during transformation. These studies will highlight the roles of CHK2 and DNAPKc in DSB repair, malignant transformation, and will shed more light on the role of DNAPKc and CHK2 in reprograming the genome. Supported by R01 ES15262 to SAB Citation Format: Ivo Teneng, Randy Willink, Kieu C. Do, Christopher Dagucon, Steven A. Belinsky. CHK2 and DNAPKc deficiency reduces DNA repair capacity and increases transformation efficiency in human bronchial epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2013-619

Chk1 suppresses bypass of mitosis and tetraploidization in p53-deficient cancer cells

Many cancer cells are unable to maintain a numerically stable chromosome complement. It is well established that aberrant cell division can generate progeny with increased ploidy, but the genetic factors required for maintenance of diploidy are not well understood. Using an isogenic model system derived by gene targeting, we examined the role of Chk1 in p53-proficient and -deficient cancer cells. Targeted inactivation of a single CHK1 allele in stably diploid cells caused an elevated frequency of mitotic bypass if p53 was naturally mutated or experimentally disrupted by homologous recombination. CHK1-haploinsufficient, p53-deficient cells frequently underwent sequential rounds of DNA synthesis without an intervening mitosis. These aberrant cell cycles resulted in whole-genome endoreduplication and tetraploidization. The unscheduled bypass of mitosis could be suppressed by targeted reversion of a p53 mutation or by exogenous expression of Cdk1. In contrast, the number of tetraploid cells was not increased in isogenic cell populations that harbor hypomorphic ATR mutations, suggesting that suppression of unscheduled mitotic bypass is a distinct function of Chk1. These results are consistent with a recently described role for Chk1 in promoting the expression of genes that promote cell cycle transitions and demonstrate how Chk1 might prevent tetraploidization during the cancer cell cycle.

Abstract 4699: A siRNA high-throughput screening identified Wee1 as determinant of Chk1 inhibitor sensitivity

Abstract Targeting Chk1 protein kinase is a valid anti cancer strategy to enhance the cytotoxic effects of radio and chemotherapy. Recent evidence disclosed a role of Chk1 in unperturbed cell proliferation and survival, implying that Chk1 inhibitors could also be effective as single agents in tumors with a specific genetic background. The effect of Chk1 inhibition on a wide panel of cancer cell lines has been herein conducted by using a specific and selective Chk1 inhibitor PF-00477736. The results showed that 2 cancer cell lines out of 16 analyzed (the ovarian cancer cell line OVCA-432 and the breast cancer cell line SK-BR3) were very sensitive to PF-00477736 treatment. To identify genes in synthetic lethality with Chk1, which may justify the sensitivity to Chk1 inhibitor of some cancer cell lines, a high-throughput screening using a siRNA library directed against 719 human protein kinases, in combination with either scramble or Chk1 siRNA, was undertaken. The ovarian cancer cell line OVCAR-5, resistant to Chk1 inhibition, was used to perform the screening. After 2 independent screenings and the validation procedure using specific esiRNAs, and the three siRNAs of each target (used in the screening as a pool), the Wee1 tyrosine kinase was identified as the most significant gene in synthetic lethality with Chk1. Treatment with both PF-00477736 and a Wee1 inhibitor (MK-1775) confirmed the remarkable synergistic effect in various human cancer cell lines (breast, ovarian, colon, prostate). Same results have been obtained with AZD-7762 Chk1 inhibitor. Isogenic cellular systems differing only for the status of p53 (the colon carcinoma cancer cell lines HCT-116 p53 +/+ and p53 -/- and the ovarian cancer cell lines A2780 and A2780/E6) showed a comparable and strong synergistic effect of the combined treatment. Interestingly, in the normal fetal human lung cell line MRC-5 the effect of the combined treatment was additive. Time lapse experiments and an evaluation of the ability of cells to form colonies, suggested that cells treated with the two inhibitors almost completely lost the ability to proliferate and failed to perform a proper cell division. A detailed molecular analysis showed that the combined treatment causes cells to undergo premature mitosis before the end of DNA replication and with damaged DNA, leading to a massive cell death by apoptosis. Taken together these preclinical data address a new promising combined targeted anticancer strategy that deserves clinical investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4699. doi:1538-7445.AM2012-4699