G2 Checkpoint Function Research Articles

Epithelial-mesenchymal transition: an organizing principle of mammalian regeneration.

Introduction: The MRL mouse strain is one of the few examples of a mammal capable of healing appendage wounds by regeneration, a process that begins with the formation of a blastema, a structure containing de-differentiating mesenchymal cells. HIF-1α expression in the nascent MRL wound site blastema is one of the earliest identified events and is sufficient to initiate the complete regenerative program. However, HIF-1α regulates many cellular processes modulating the expression of hundreds of genes. A later signal event is the absence of a functional G1 checkpoint, leading to G2 cell cycle arrest with increased cellular DNA but little cell division observed in the blastema. This lack of mitosis in MRL blastema cells is also a hallmark of regeneration in classical invertebrate and vertebrate regenerators such as planaria, hydra, and newt. Results and discussion: Here, we explore the cellular events occurring between HIF-1α upregulation and its regulation of the genes involved in G2 arrest (EVI-5, γH3, Wnt5a, and ROR2), and identify epithelial-mesenchymal transition (EMT) (Twist and Slug) and chromatin remodeling (EZH-2 and H3K27me3) as key intermediary processes. The locus of these cellular events is highly regionalized within the blastema, occurring in the same cells as determined by double staining by immunohistochemistry and FACS analysis, and appears as EMT and chromatin remodeling, followed by G2 arrest determined by kinetic expression studies.

Preclinical evaluation of SC0191, a small molecule inhibitor of Wee1 kinase.

e15637 Background: TP53 mutation is common in cancer cells, especially in small cell lung cancer (SCLC) and pancreatic cancer (PC). The G1 checkpoint function is lost in these cancer cells, which more rely on G2 checkpoint to repair damaged DNA. Wee1 is a serine/threonine kinase, which phosphorylates the Tyr15 locus of CDC2, inhibits the activity of CDC2, and stays in the G2 phase. Wee1 inhibition can promote the cells with DNA damage into the M phase, thus driving the TP53 mutant cancer cells to apoptosis. The purpose of this study was to investigate the in vitro and in vivo antitumor activity of SC0191, a small molecule inhibitor of Wee1 kinase, in preclinical models of SCLC and PC with TP53 mutation. Methods: The kinase inhibiting activity of SC0191 was determined using the Wee1 kinase assays. The cellular anti-proliferative activity was evaluated with TP53 mutant NCI-H446 SCLC and BxPC-3 PC cell lines. The in vivo antitumor activity of SC0191 was evaluated in the NCI-H446 and BxPC-3 cells-derived xenograft (CDX) mouse models, respectively. Results: SC0191 displayed potent kinase inhibiting activity for Wee1 with IC50 22.3 nM. SC0191 significantly inhibited tumor cell proliferation in TP53 mutant NCI-H446 cells with IC50 231.1 nM and BxPC-3 cells with IC50 223.8 nM. Moreover, SC0191 oral administration showed significant antitumor efficacy which was better than AZD1775, in the NCI-H446 and BxPC-3 CDX mouse models, respectively. Conclusions: A novel Wee1 inhibitor, SC0191, has demonstrated excellent antitumor efficacy in TP53 mutant solid cancer preclinical studies, and represents a promising clinical candidate for treating solid tumors, such as SCLC and PC.

PO-021 Inhibition of MK2 stabilises wild-type P53 and improves temozolomide efficacy in glioblastoma

Introduction Temozolomide (TMZ) remains the first line therapy for glioblastoma since its approval in 2005. TMZ efficacy is marginal as patients succumb to the disease within two years, urging the need for new therapeutic approaches. Frequent genetic alterations in the DNA damage machinery of most cancer cells opens a window of possible therapeutic vulnerability. Tumours lacking p53 rely heavily on the G2/M cell cycle checkpoint for DNA repair, due to the loss of a functional G1 checkpoint. Therefore, inhibiting the G2/M checkpoint allows for synthetic lethality, as these cells are forced to continue through the cycle with unrepaired DNA ultimately resulting in cell death. It was previously reported that in the absence of p53, cells depend on the checkpoint kinase MK2 for cell cycle arrest and survival after DNA damage by doxorubicin. However, it is currently unknown if targeting MK2 is a viable therapeutic approach for glioblastoma therapy in conjunction with TMZ. Thus, we aimed to investigate the effect of inhibiting MK2 and whether it will improve the effectiveness of TMZ in glioblastoma. Material and methods Inhibition of MK2 was conducted using siRNA, CRISPR-Cas9 and pharmacological inhibitors. Signalling pathways, stability and nuclear retention of proteins was determined by Western blotting. Cell cycle arrest was analysed using FUCCI transfected cells and flow cytometry. Cellular senescence was visualised by staining the presence of b-galactosidase activity. Glioblastoma cell survival was assessed by colony formation and 3D spheroid tumour growth. Results and discussions Our data demonstrates that inhibition of MK2 increases the stability and expression of p53, indirectly by reducing the phosphorylation of its ligase MDM2. This suggests that MK2 plays a role in regulating p53, as inhibition of MK2 increased the levels of active p53 inside the nucleus. We also found that the combination of TMZ and MK2 inhibition did not induce G2/M arrest as expected, but G0/G1 arrest. This increase of cells in G0/G1 led to cellular senescence through the reactivation of the p53-p21 pathway. Moreover, we found that MK2 inhibition enhances the efficacy of TMZ by attenuating long-term clonal and 3D spheroid growth in p53 proficient cells, unlike previously described. Conclusion The combination of MK2 inhibition alongside TMZ provides a new therapeutic strategy to enhance the effectiveness of current chemotherapy in p53 proficient tumours.

Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies.

Radiotherapy has been accounted as the most comprehensive cancer treatment modality over the past few decades. However, failure of this treatment modality occurs in several malignancies due to the resistance of cancer cells to radiation. It was previously reported by the present authors that defective cell cycle checkpoints could be used as biomarkers for predicting the responsiveness to radiation in individual patients with cholangiocarcinoma (CCA). However, identification of functional defective cell cycle checkpoints from cells from a patient's tissues is cumbersome and not applicable in the clinic. The present study evaluated the radiosensitization potential of etoposide in p53-defective CCA KKU-M055 and KKU-M214 cell lines. Treatment with etoposide enhanced the responsiveness of two p53-defective CCA cell lines to radiation independent of G2 checkpoint function. In addition, etoposide treatment increased radiation-induced cell death without altering the dominant mode of cell death of the two cell lines. These findings indicate that etoposide could be used as a radiation sensitizer for p53-defective tumors, independent of the function of G2 checkpoint.

Distinct Cell-Cycle Control in Two Different States of Mouse Pluripotency

SummaryMouse embryonic stem cells (ESCs) cultured in serum are characterized by hyper-phosphorylated RB protein, lack of G1 control, and rapid progression through the cell cycle. Here, we show that ESCs grown in the presence of two small-molecule inhibitors (2i ESCs) have a longer G1-phase with hypo-phosphorylated RB, implying that they have a functional G1 checkpoint. Deletion of RB, P107, and P130 in 2i ESCs results in a G1-phase similar to that of serum ESCs. Inhibition of the ERK signaling pathway in serum ESCs results in the appearance of hypo-phosphorylated RB and the reinstatement of a G1 checkpoint. In addition, induction of a dormant state by the inhibition of MYC, resembling diapause, requires the presence of the RB family proteins. Collectively, our data show that RB-dependent G1 restriction point signaling is active in mouse ESCs grown in 2i but abrogated in serum by ERK-dependent phosphorylation.

Abstract LB-319: Development and characterization of ADC999: A novel, potent orally available Wee1 inhibitor with robust antitumor efficacy in vivo

Abstract Wee1 is a tyrosine kinase that phosphorylates cyclin-dependent kinase 1 (CDC2) to inactivate the CDC2/cyclinB complex. Wee1 is therefore a key regulator of the cell cycle, maintaining the G2 checkpoint arrest to allow for pre-mitotic repair upon genomic stress. Unlike normal cells, most p53-deficient tumors lack a functional G1 checkpoint and instead rely on the G2 checkpoint for DNA repair. Abrogation of the G2 checkpoint by a Wee1 inhibitor has been shown to sensitize p53-deficient tumors to a variety of DNA-damaging anticancer agents. Strong chemosensitization has also been demonstrated in multiple pre-clinical models using targeted agents including CHK1 or PARP inhibitors. These studies combined with recent clinical data demonstrating robust anti-tumor activity in combination with carboplatin or as a single agent in tumors under high replicative stress make Wee1 a promising target for anticancer therapy. With only one inhibitor (AZD1775) in clinical development and very few reports of compounds at the preclinical stage, the development of alternative Wee1 inhibitors is of significant medical relevance. In this study, we describe the development of a novel and highly potent small molecule inhibitor of Wee1 (ADC999) and subsequent analogs. These inhibitors exhibit single digit nM IC50 values against Wee1 and good overall selectivity profiles against the kinome. In cells, target engagement was demonstrated through the inhibition of both CDC2- and CDK2-dependent Wee1 phosphorylation. In line with the mechanism of action, γH2AX and apoptosis induction was also observed in a dose-dependent manner. Further profiling in a panel of cell lines indicated strong anti-proliferative activity both in combination with cytotoxics and in monotherapy. Single agent activity was observed in multiple cancer types, including gynecological, lung and breast. In vivo, oral administration of ADC999 as a single agent led to excellent antitumor activity in a KRAS-mutant non-small cell lung carcinoma xenograft model. Responses ranged from dose-dependent tumor growth inhibition to complete and sustained regressions. ADC999 was well tolerated following a daily dosing regimen and over a prolonged period with no adverse effects observed. Evaluation of ADC999 anti-tumor activity will also be discussed in a panel of genetically-characterized patient-derived xenografts (PDXs) both as monotherapy (mini-trial design) and in combination with PARP inhibitors. In summary, we describe the development and profiling of a novel, highly potent orally available Wee1 inhibitor with robust antitumor efficacy in vivo. This inhibitor and potential back-up compounds will provide new opportunities to fully exploit the therapeutic potential of Wee1 inhibition both as standalone intervention and in combination modalities. Citation Format: Gerald Gavory, Colin O'Dowd, Ewelina Rozycka, Caroline Boyd, Beronia Gorges, Estelle McLean, Julien Daubriac, Shane Rountree, Steven Shepherd, Stephanie Burton, Mary McFarland, Adam Treder, Andy Wilkinson, Frank Burkamp, Tim Harrison. Development and characterization of ADC999: A novel, potent orally available Wee1 inhibitor with robust antitumor efficacy in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-319. doi:10.1158/1538-7445.AM2017-LB-319

Abstract B17: Development of novel, potent orally available Wee1 inhibitors with robust antitumor efficacy in vivo

Abstract Wee1 is a tyrosine kinase that phosphorylates cyclin-dependent kinase 1 (CDC2) to inactive the CDC2/cyclinB complex. Wee1 is therefore a key regulator of the cell cycle, maintaining the G2 checkpoint arrest to allow for pre-mitotic repair upon genomic stress. Unlike normal cells, most p53-deficient tumors lack a functional G1 checkpoint and instead rely on the G2 checkpoint for DNA repair. Abrogation of the G2 checkpoint by a Wee1 inhibitor therefore sensitizes p53-deficient tumors to DNA-damaging anticancer agents and enhances their cytotoxic effect. Strong evidence for chemo-sensitization has been demonstrated in multiple pre-clinical models and also in clinical trials. These clinical observations combined with recent reports demonstrating single agent efficacy make Wee1 a promising target for anticancer therapy. With only one inhibitor (AZD1775) in clinical development and very few reports of compounds at the preclinical stage, the development of alternative Wee1 inhibitors is of significant medical relevance. In this study, we describe the development of novel and highly potent small molecule inhibitors of Wee1 emanating from 2 distinct chemical series (ADC730, ADC999). These inhibitors exhibit single digit nM IC50 values versus the enzyme and good selectivity profiles against the kinome. In cells, target engagement was demonstrated through the inhibition of both CDC2- and CDK2-dependent Wee1 phosphorylation. In line with the mechanism of action, gH2AX and apoptosis induction was also observed in a dose-dependent manner. Further profiling in panels of cell lines indicated strong anti-proliferative activity both in combination with cytotoxics (i.e. gemcitabine) and in monotherapy. Single agent activity was observed in multiple cancer types, including gynecological, lung, colorectal and breast. In vivo, oral administration of ADC730 in combination with gemcitabine resulted in significant dose-dependent growth reduction in a HT29 tumor xenograft model and the combination was synergistic. Dosed orally and as single agents, both ADC730 and ADC999 also demonstrated excellent antitumor activity in the A427 KRAS-mutant lung carcinoma xenograft model. Responses ranged from dose-dependent tumor growth inhibition to complete regressions. ADC730 and ADC999 were well tolerated in these studies with no signs of adverse effects observed. In summary, we describe the development and profiling of novel, highly potent orally available Wee1 inhibitors with robust antitumor efficacy in vivo. These inhibitors will provide new opportunities to fully exploit the therapeutic potential of Wee1 inhibition, either as standalone intervention or in combination modalities. Citation Format: Gerald Gavory, Colin O'Dowd, Ewelina Rozycka, Caroline Boyd, Beronia Gorges, Estelle McLean, Shane Rountree, Steven Sheperd, Stephanie Burton, Mary McFarland, Dominic Janssen, Adam Treder, Andy Wilkinson, Frank Burkamp, Tim Harrison. Development of novel, potent orally available Wee1 inhibitors with robust antitumor efficacy in vivo [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B17.

Abstract LB-159: A novel, potent and selective inhibitor of Wee1 with robust antitumor activity in various cancer xenograph models

Abstract Wee1 is a tyrosine kinase that phosphorylates cyclin-dependent kinase 1 (CDK1/CDC2) to inactive the CDC2/cyclinB complex. Wee1 is therefore a key regulator of the cell cycle, maintaining the G2 checkpoint arrest to allow for premitotic repair in case of DNA damage. Unlike normal cells, most p53-deficient tumors lack a functional G1 checkpoint and instead rely on the G2 checkpoint for DNA repair. Abrogation of the G2 checkpoint by a Wee1 inhibitor may therefore sensitize p53-deficient tumors to DNA-damaging anticancer agents and enhance their cytotoxic effect. Strong evidence for chemo-sensitization has been demonstrated from multiple pre-clinical models and also from clinical trials. In particular, treatment in combination with carboplatin recently showed encouraging antitumor activity in patients with p53-mutated ovarian cancer refractory or resistant to standard first-line therapy. These clinical observations combined with recent reports demonstrating single agent efficacy in specific contexts make Wee1 a promising target for anticancer therapy. With only one inhibitor in clinical development (AZD1775) and very limited reports at the preclinical stage, the development of alternative, novel Wee1 inhibitors may have significant therapeutic value. In this study, we describe the development and characterization of ADC730, a potent and highly selective small molecule Wee1 inhibitor. ADC730 inhibits Wee1 kinase activity with an IC50 of <5 nM and demonstrates excellent selectivity when profiled against a diverse panel of kinases. In cellular assays, ADC730 potently inhibited CDC2-dependent Wee1 phosphorylation. Further studies in a panel of cancer cell lines demonstrated strong anti-proliferative activity of ADC730 both in combination with gemcitabine or in monotherapy modalities. Single agent activity was observed in multiple cancer types including lung and kidney. gH2AX foci formation followed by apoptosis induction were typically observed in a dose-dependent manner for the most sensitive cell lines. In vivo, oral administration of ADC730 (10, 30 mg/kg/day) in combination with gemcitabine caused a significant dose-dependent growth reduction of HT29 tumors and the combination was synergistic. ADC730 was well tolerated in all cases with no signs of adverse effects. Dosed orally as a single agent (10, 30 mg/kg/day), ADC730 also demonstrated robust dose-dependent anti-tumor efficacy in the A427 lung carcinoma model with inhibition of tumor growth consistent with the pharmacodynamic modulation of Wee1 signaling. In comparison and under similar experimental conditions, AZD1775 demonstrated similar efficacy profiles at significantly higher doses. To conclude, we describe the development and profiling towards candidate nomination of ADC730, a novel potent highly selective Wee1 inhibitor with robust efficacy in cancer xenograft models both in combination and single agent modalities. Subsequent analogs and examples with potentially enhanced profiles will also be described. Citation Format: Gerald Gavory, Colin O’Dowd, Frank Burkamp, Shane Rountree, Ewelina Rozycka, Caroline Boyd, Beronia Gorges, Stephanie Burton, Andy Wilkinson, Steven Shepherd, Dominic Janssen, Mary McFarland, Shane Rountree, Tim Harrison. A novel, potent and selective inhibitor of Wee1 with robust antitumor activity in various cancer xenograph models. [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 LB-159.

Extremely stringent activation of p16INK4a prevents immortalization of uterine cervical epithelial cells without human papillomavirus oncogene expression.

Cervical epithelial cell immortalization with defined genetic factors without viral oncogenes has never been reported. Here we report that HPV-negative cervical epithelial cells failed to be immortalized by telomerase activation or the combination of p53 knockdown and telomerase activation. Under those conditions, p16INK4a expression was always elevated during the late stage of limited cell lifespan, suggesting that cervical epithelial cells possess an intrinsic property of uniquely stringent activation of p16INK4a, which may offer an explanation for the rarity of HPV-negative cervical cancer. Combining p16INK4a knockdown with telomerase activation resulted in efficient immortalization of HPV-negative cervical epithelial cells under ordinary culture conditions. Compared with the HPV16-E6E7-immortalized cell lines derived from the same primary cell sources, the novel HPV-negative immortalized cell lines had lower degrees of chromosomal instability, maintained more sensitive p53/p21 response to DNA damage, exhibited more stringent G2 checkpoint function, and were more resistant to replication-stress-induced genomic instability. The newly immortalized HPV-negative cervical epithelial cell lines were non-tumorigenic in nude mice. The cell lines can be used not only as much-needed HPV-negative non-malignant cell models but also as starting models that can be genetically manipulated in a stepwise fashion to investigate the roles of defined genetic alterations in the development of HPV-negative cervical cancer.

Evaluation of Methods for Gene Selection in Melanoma Cell Lines

A major objective in microarray experiments is to identify a panel of genes that are associated with a disease outcome or trait. Many statistical methods have been proposed for gene selection within the last fifteen years. While the comparison of some of these methods has been done, most of them concentrated on finding gene signatures based on two groups. This study evaluates four gene selection methods when the outcome of interested is continuous in nature. We provide a comparative review of four methods: the Statistical Analysis of Microarrays (SAM), the Linear Models for Microarray Analysis (LIMMA), the Lassoed Principal Components (LPC), and the Quantitative Trait Analysis (QTA). Comparison is based on the power to identify differentially expressed genes, the predictive ability of the genelists for a continuous outcome (G2 checkpoint function), and the prognostic properties of the genelists for distant metastasis-free survival. A simulated dataset and a publicly available melanoma cell lines dataset are used for simulations and validation, respectively. A primary melanoma dataset is used for assessment of prognosis. No common genes were found among the genelists from the four methods. While the SAM was generally the best in terms of power, the QTA genelist performed the best in the prediction of the G2 checkpoint function. Identification of genelists depends on the choice of the gene selection method. The QTA method would be preferred over the other approaches in predicting a quantitative outcome in melanoma research. We recommend the development of more robust statistical methods for differential gene expression analysis.

Mechanisms of chromosomal instability in melanoma

A systems biology approach was applied to investigate the mechanisms of chromosomal instability in melanoma cell lines. Chromosomal instability was quantified using array comparative genomic hybridization to identify somatic copy number alterations (deletions and duplications). Primary human melanocytes displayed an average of 8.5 alterations per cell primarily representing known polymorphisms. Melanoma cell lines displayed 25 to 131 alterations per cell, with an average of 68, indicative of chromosomal instability. Copy number alterations included approximately equal numbers of deletions and duplications with greater numbers of hemizygous (-1,+1) alterations than homozygous (-2,+2). Melanoma oncogenes, such as BRAF and MITF, and tumor suppressor genes, such as CDKN2A/B and PTEN, were included in these alterations. Duplications and deletions were functional as there were significant correlations between DNA copy number and mRNA expression for these genes. Spectral karyotype analysis of three lines confirmed extensive chromosomal instability with polyploidy, aneuploidy, deletions, duplications, and chromosome rearrangements. Bioinformatic analysis identified a signature of gene expression that was correlated with chromosomal instability but this signature provided no clues to the mechanisms of instability. The signature failed to generate a significant (P = 0.105) prediction of melanoma progression in a separate dataset. Chromosomal instability was not correlated with elements of DNA damage response (DDR) such as radiosensitivity, nucleotide excision repair, expression of the DDR biomarkers γH2AX and P-CHEK2, nor G1 or G2 checkpoint function. Chromosomal instability in melanoma cell lines appears to influence gene function but it is not simply explained by alterations in the system of DDR.

A prognostic signature of G₂ checkpoint function in melanoma cell lines

As DNA damage checkpoints are barriers to carcinogenesis, G2 checkpoint function was quantified to test for override of this checkpoint during melanomagenesis. Primary melanocytes displayed an effective G2 checkpoint response to ionizing radiation (IR)-induced DNA damage. Thirty-seven percent of melanoma cell lines displayed a significant defect in G2 checkpoint function. Checkpoint function was melanoma subtype-specific with “epithelial-like” melanoma lines, with wild type NRAS and BRAF displaying an effective checkpoint, while lines with mutant NRAS and BRAF displayed defective checkpoint function. Expression of oncogenic B-Raf in a checkpoint-effective melanoma attenuated G2 checkpoint function significantly but modestly. Other alterations must be needed to produce the severe attenuation of G2 checkpoint function seen in some BRAF-mutant melanoma lines. Quantitative trait analysis tools identified mRNA species whose expression was correlated with G2 checkpoint function in the melanoma lines. A 165 gene signature was identified with a high correlation with checkpoint function (p < 0.004) and low false discovery rate (≤ 0.077). The G2 checkpoint gene signature predicted G2 checkpoint function with 77–94% accuracy. The signature was enriched in lysosomal genes and contained numerous genes that are associated with regulation of chromatin structure and cell cycle progression. The core machinery of the cell cycle was not altered in checkpoint-defective lines but rather numerous mediators of core machinery function were. When applied to an independent series of primary melanomas, the predictive G2 checkpoint signature was prognostic of distant metastasis-free survival. These results emphasize the value of expression profiling of primary melanomas for understanding melanoma biology and disease prognosis.

A prognostic signature of G₂ checkpoint function in melanoma cell lines

A prognostic signature of G₂ checkpoint function in melanoma cell lines

A predictive mathematical model of the DNA damage G2 checkpoint

A predictive mathematical model of the transition from the G2 phase in the cell cycle to mitosis (M) was constructed from the known interactions of the proteins that are thought to play significant roles in the G2 to M transition as well as the DNA damage- induced G2 checkpoint. The model simulates the accumulation of active cyclin B1/Cdk1 (MPF) complexes in the nucleus to activate mitosis, the inhibition of this process by DNA damage, and transport of component proteins between cytoplasm and nucleus. Interactions in the model are based on activities of individual phospho-epitopes and binding sites of proteins involved in G2/M. Because tracking phosphoforms leads to combinatorial explosion, we employ a rule-based approach using the BioNetGen software. The model was used to determine the effects of depletion or over-expression of selected proteins involved in the regulation of the G2 to M transition in the presence and absence of DNA damage. Depletion of Plk1 delayed mitotic entry and recovery from the DNA damage-induced G2 arrest and over-expression of MPF attenuated the DNA damage-induced G2 delay. The model recapitulates the G2 delay observed in the biological response to varying levels of a DNA damage signal. The model produced the novel prediction that depletion of pkMyt1 results in an abnormal biological state in which G2 cells with DNA damage accumulate inactive nuclear MPF. Such a detailed model may prove useful for predicting DNA damage G2 checkpoint function in cancer and, therefore, sensitivity to cancer therapy.

A prognostic signature of defective p53-dependent G1 checkpoint function in melanoma cell lines

Melanoma cell lines and normal human melanocytes (NHM) were assayed for p53-dependent G1 checkpoint response to ionizing radiation (IR)-induced DNA damage. Sixty-six percent of melanoma cell lines displayed a defective G1 checkpoint. Checkpoint function was correlated with sensitivity to IR with checkpoint-defective lines being radio-resistant. Microarray analysis identified 316 probes whose expression was correlated with G1 checkpoint function in melanoma lines (P≤0.007) including p53 transactivation targets CDKN1A, DDB2, and RRM2B. The 316 probe list predicted G1 checkpoint function of the melanoma lines with 86% accuracy using a binary analysis and 91% accuracy using a continuous analysis. When applied to microarray data from primary melanomas, the 316 probe list was prognostic of 4-yr distant metastasis-free survival. Thus, p53 function, radio-sensitivity, and metastatic spread may be estimated in melanomas from a signature of gene expression.

A prognostic signature of defective p53-dependent G1 checkpoint function in melanoma cell lines: A signature of defective p53 function in melanoma

A prognostic signature of defective p53-dependent G1 checkpoint function in melanoma cell lines: A signature of defective p53 function in melanoma

Can an iPS cell secure its genomic fidelity?

The hallmark of stem cells is their capacity to maintain a stringent mechanism to ensure their genomic integrity. This is particularly crucial for embryonic stem (ES) cells, which hold the potential to give rise to an entire organism, including its germ cells. In contrast to mutations occurring in mature somatic cells, mutations in ES cells may be endowed to various cell types in the organism, and may be passed on to its progeny. Indeed, mutation frequency in ES cells is much lower compared with somatic cells.1 To achieve this, ES cells are extremely sensitive to DNA damage and they readily undergo apoptosis or differentiation, resulting in sequestering of damaged cells from the pluripotent pool. ES cells lack a functional G1 checkpoint, enabling newly formed cells with DNA damage to enter another phase of DNA replication. Escalating the DNA damage leads to mutational burden and promotes apoptosis. In addition, ES cells differ from somatic cells in their capacity to repair damaged DNA. Somatic cells predominantly utilize error-prone nonhomologous end joining, which may promote accumulation of mutations, whereas ES cells predominantly utilized the high-fidelity homology-mediated repair pathway, thereby minimizing the incurrence of mutations during the repair process.2, 3

Topoisomerase IIα maintains genomic stability through decatenation G2 checkpoint signaling

Topoisomerase IIα (topoIIα) is an essential mammalian enzyme that topologically modifies DNA and is required for chromosome segregation during mitosis. Previous research suggests that inhibition of topoII decatenatory activity triggers a G2 checkpoint response, which delays mitotic entry due to insufficient decatenation of daughter chromatids. Here we examine the effects of both topoIIα and topoIIβ on decatenatory activity in cell extracts, DNA damage and decatenation G2 checkpoint function, and the frequencies of p16INK4A allele loss and gain. In diploid human fibroblast lines, depletion of topoIIα by siRNA was associated with severely reduced decatenatory activity, delayed progression from G2 into mitosis, and insensitivity to G2 arrest induced by the topoII catalytic inhibitor ICRF-193. Furthermore, interphase nuclei of topoIIα-depleted cells displayed increased frequencies of losses and gains of the tumor suppressor genetic locus p16INK4A. This study demonstrates that the topoIIα protein is required for decatenation G2 checkpoint function, and inactivation of decatenation and the decatenation G2 checkpoint leads to abnormal chromosome segregation and genomic instability.

Revised genetic requirements for the decatenation G2 checkpoint: The role of ATM

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.

Abstract C207: Checkpoint abrogation and potentiation of cytotoxic chemotherapeutics with a novel checkpoint kinase 1 inhibitor

Abstract Conventional chemotherapeutic agents such as gemcitabine, cisplatin or irinotecan induce DNA damage and activate cell cycle checkpoints. P53 defective tumors lack a functional G1 checkpoint and rely heavily on the S and G2 checkpoints, and the effector kinase Chk1, for protection against this DNA damage. Inhibiting Chk1 potentiates the anti-tumor effects of these cytotoxic chemotherapeutic agents. Targeting Chk1 is a potential therapeutic opportunity for potentiating the anti-tumor efficacy of DNA damaging cytotoxic chemotherapeutic drugs without increasing their toxicity to normal cells. Elaboration of a designed kinase directed fragment core utilizing X-ray structure guided design lead to the identification of a potent pyridone series of Chk1 inhibitors. Further profiling identified V158411 as the lead candidate. X-ray crystallography identified V158411 as being bound to the ATPase site in the kinase domain of Chk1. V158411 potently inhibited Chk1 and Chk2 with IC50s of 4.4 and 4.5nM respectively. The addition of V158411 to gemcitabine or camptothecin treated cells abrogated the cell cycle checkpoints induced by these agents resulting in the expected modulation of cell cycle proteins and increased apoptosis. V158411 potentiated the cytotoxicity of gemcitabine, cisplatin, SN38 and camptothecin in a variety of p53 deficient but not proficient human tumor cell lines in vitro. V158411 could be formulated in a simple aqueous form suitable for i.v. dosing. In nude mice, V158411 was well tolerated as a single agent (MTD &amp;gt;100mg/kg) and in combination with irinotecan. Intravenous administration to rats and mice resulted in low plasma clearances (20mL/min/kg) and long half-lives (2.9–3.7h). In tumor bearing animals, V158411 was detected at high concentrations in the tumor (tumor:plasma AUC ratio of 4.7) with a long tumor elimination half life of 22 hours. No pharmacologically relevant in vivo drug-drug interaction with irinotecan was identified. V158411 potentiated the anti-tumor activity of gemcitabine and irinotecan in a variety of human tumor xenograft models without additional systemic toxicity. These results demonstrate the potential of combining V158411 with standard of care chemotherapeutic agents to potentiate the therapeutic efficacy of these agents without increasing their toxicity to normal cells. Based on this data, the clinical development of V158411 is currently being actively pursued. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C207.