Common fragile sites

Common fragile sites are regions showing site-specific gaps and breaks on metaphase chromosomes after partial inhibition of DNA synthesis. Common fragile sites are normally stable in somatic cells. However, following treatment of cultured cells with replication inhibitors, fragile sites display gaps, breaks, rearrangements and other features of unstable DNA. Studies showing that fragile sites and associated genes are frequently deleted or rearranged in many cancer cells have clearly demonstrated their importance in genome instability in cancer. Until recently, little was known about the molecular nature and mechanisms involved in fragile site instability. From studies conducted in many laboratories, it is now known that fragile sites extend over large regions, are associated with genes, exhibit delayed replication, and contain regions of high DNA flexibility. Recent findings from our laboratory showing that the key cell cycle checkpoint genes are important for genome stability at fragile sties have shed new light on these mechanisms and on the significance of these sites in cancer and normal chromosome structure. Since their discovery over two decades ago, much has been learned regarding their significance in chromosome structure and instability in cancer, but a number of key questions remain, including why these sites are 'fragile' and the impact of this instability on associated genes in cancer cells. These and other questions have been addressed by participants of this meeting, which highlighted instability at common fragile sites. This brief review is intended to provide background on common fragile sites that has led up to many of the studies presented in the accompanying reports in this volume and not to summarize the findings presented therein. Some aspects of this review were taken from Glover et al. (T.W. Glover, M.F. Arlt, A.M. Casper, S.G. Durkin, Mechanisms of common fragile site instability, Hum. Molec. Genet. 14 (in press). [1]).     

RRM1 maintains centrosomal integrity via CHK1 and CDK1 signaling during replication stress

DNA lesion-induced centrosomal abnormalities during the replication phase are relatively unknown. Here, we report that RNAi-mediated depletion of RRM1 induces cell-cycle arrest at the replication phase, along with severe DNA damage and centrosomal amplification. Interestingly, CHK1 depletion synergistically increased RRM1-depletion-induced centrosomal amplification. In response to hydroxyurea, CHK1 was delocalized from the centrosome by RRM1 depletion. Moreover, CDK1, which functions in centrosome separation and is inhibited by CHK1, was found to be essential for RRMI1-depletion-induced centrosomal amplification. Thus, we herein demonstrate that RRM1 preserves chromosomal stability via the CHK1- and CDK1-dependent stabilization of the centrosomal integrity at the replication stage.     

Histological type-selective, tumor-predominant expression of a novel CHK1 isoform and infrequent in vivo somatic CHK2 mutation in small cell lung cancer

Inactivation of p53, which represents the most prevalent genetic alteration in lung cancer, has been shown to play a crucial role in the acquisition of genomic instability. We examined 44 lung cancer specimens to search for mutations in the CHK1 and CHK2 genes, which have been suggested to play roles in regulating p53 after DNA damage. We found that the CHK2 gene was somatically mutated in lung cancer in vivo, although at a low frequency, and that a previously undescribed shorter isoform of CHK1 was expressed preferentially in small cell lung cancer in a tumor-predominant manner. Additional studies are warranted to investigate the functional significance of these changes as well as the potential involvement of other components in this important pathway to maintain genomic stability.     

Interplay between ATM and ATR in the regulation of common fragile site stability

Common fragile sites are specific genomic loci that form constrictions and gaps on metaphase chromosomes under conditions that slow, but do not arrest, DNA replication. These sites have been shown to have a role in various chromosomal rearrangements in tumors. Different DNA damage response proteins were shown to regulate fragile site stability, including ataxia-telangiectasia and Rad3-related (ATR) and its effector Chk1. Here, we investigated the role of ataxia-telangiectasia mutated (ATM), the main transducer of DNA double-strand break (DSB) signal, in this regulation. We demonstrate that replication stress conditions, which induce fragile site expression, lead to DNA fragmentation and recruitment of phosphorylated ATM to nuclear foci at DSBs. We further show that ATM plays a role in maintaining fragile site stability, which is revealed only in the absence of ATR. However, the activation of ATM under these replication stress conditions is ATR independent. Following conditions that induce fragile site expression both ATR and ATM phosphorylate Chk1, suggesting that both proteins regulate fragile site expression probably via their effect on Chk1 activation. Our findings provide new insights into the interplay between ATR and ATM pathways in response to partial replication inhibition and in the regulation of fragile site stability.     

Efficacy of CHK inhibitors as single agents in MYC-driven lymphoma cells

CHK1 and CHK2 function as effectors of cell cycle checkpoint arrest following DNA damage. Small molecule inhibitors of CHK proteins are under clinical evaluation in combination with chemotherapeutic agents known to induce DNA damage. We examined whether CHK inhibitors could be effective as single agents in malignant cells with inherent DNA damage because of deregulated expression of the oncogene c-Myc. Eμ-myc lymphoma cells showed a dramatic increase in the extent of DNA damage and DNA damage response (DDR) signalling within 1 h of treatment with CHK1 inhibitors followed by caspase-dependent apoptosis and cell death. In p53 wild-type/ARF null Eμ-myc lymphoma cells, apoptotic cell death was preceded by accumulation of DNA damage and the amount of DNA damage correlated with the extent of cell death. This effect was not observed in normal B cells indicating that DNA damage accumulation following CHK inhibition was specific to Eμ-myc lymphoma cells that exhibit inherent DNA damage because of MYC-induced replication stress. Similar results were obtained with another structurally distinct CHK-inhibitor. Eμ-myc p53 null lymphoma cells were more sensitive to a dual CHK1/CHK2 inhibitor than to a CHK1-specific inhibitor. In all cases, the level of DNA damage following treatment was the most consistent indicator of drug sensitivity. Our results suggest that CHK inhibitors would be beneficial therapeutic agents in MYC-driven cancers. We propose that inhibitors of CHK can act in a synthetically lethal manner in cancers with replication stress as a result of these cancers being reliant on CHK proteins for an effective DDR and cell survival.     

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.     

Mutation analysis of the CHK2 gene in breast carcinoma and other cancers

Background

Mutations in the CHK2 gene at chromosome 22q12.1 have been reported in families with Li-Fraumeni syndrome. Chk2 is an effector kinase that is activated in response to DNA damage and is involved in cell-cycle pathways and p53 pathways.

Methods

We screened 139 breast tumors for loss of heterozygosity at chromosome 22q, using seven microsatellite markers, and screened 119 breast tumors with single-strand conformation polymorphism and DNA sequencing for mutations in the CHK2 gene.

Results

Seventy-four of 139 sporadic breast tumors (53%) show loss of heterozygosity with at least one marker. These samples and 45 tumors from individuals carrying the BRCA2 999del5 mutation were screened for mutations in the CHK2 gene. In addition to putative polymorphic regions in short mononucleotide repeats in a non-coding exon and intron 2, a germ line variant (T59K) in the first coding exon was detected. On screening 1172 cancer patients for the T59K sequence variant, it was detected in a total of four breast-cancer patients, two colon-cancer patients, one stomach-cancer patient and one ovary-cancer patient, but not in 452 healthy individuals. A tumor-specific 5' splice site mutation at site +3 in intron 8 (TTgt [a → c]atg) was also detected.

Conclusion

We conclude that somatic CHK2 mutations are rare in breast cancer, but our results suggest a tumor suppressor function for CHK2 in a small proportion of breast tumors. Furthermore, our results suggest that the T59K CHK2 sequence variant is a low-penetrance allele with respect to tumor growth.     

The fragile histidine triad/common chromosome fragile site 3B locus and repair-deficient cancers

In various studies of sporadic breast cancers, 40-70% were strongly positive for fragile histidine triad (Fhit) protein expression, whereas only 18% of BRCA2 mutant breast cancers demonstrated strong Fhit expression, suggesting that the BRCA2 repair function may be necessary to retain intact fragile common chromosome fragile site 3B(FRA3B)/FHITloci. In the current study, 22 breast tumors with deleterious BRCA1 mutations were analyzed for Fhit expression by immunohistochemistry in a case-control matched pair analysis. Loss of Fhit expression was significantly more frequent in the BRCA1 cancers compared with sporadic breast tumors (9% Fhit positive versus 68% Fhit positive), suggesting that the BRCA1 pathway is also important in protecting the FRA3B/FHIT locus from damage. To investigate the relationship between repair gene deficiencies and induction of chromosome fragile sites in vitro, we have analyzed the frequency of aphidicolin induction of chromosome gaps and breaks in PMS2-, BRCA1-, MSH2-, MLH1-, FHIT-, and TP53-deficient cell lines. Each of the repair-deficient cell lines showed elevated expression of chromosome gaps and breaks, consistent with the proposal that proteins involved in mismatch and double-strand break repair are important in maintaining the integrity of common fragile regions. Correspondingly, genes at common fragile sites may sustain elevated levels of DNA damage in cells with deficient DNA repair proteins such as those mutated in several familial cancer syndromes.     

PARP and CHK inhibitors interact to cause DNA damage and cell death in mammary carcinoma cells

The present studies examined viability and DNA damage levels in mammary carcinoma cells following PARP1 and CHK1 inhibitor drug combination exposure. PARP1 inhibitors [AZD2281 ; ABT888 ; NU1025 ; {"type":"entrez-nucleotide","attrs":{"text":"AG014699","term_id":"3649917","term_text":"AG014699"}}AG014699] interacted with CHK1 inhibitors [UCN-01 ; AZD7762 ; LY2603618] to kill mammary carcinoma cells. PARP1 and CHK1 inhibitors interacted to increase both single strand and double strand DNA breaks that correlated with increased γH2AX phosphorylation. Treatment of cells with CHK1 inhibitors increased the phosphorylation of CHK1 and ERK1/2. Knock down of ATM suppressed the drug-induced increases in CHK1 and ERK1/2 phosphorylation and enhanced tumor cell killing by PARP1 and CHK1 inhibitors. Expression of dominant negative MEK1 enhanced drug-induced DNA damage whereas expression of activated MEK1 suppressed both the DNA damage response and tumor cell killing. Collectively our data demonstrate that PARP1 and CHK1 inhibitors interact to kill mammary carcinoma cells and that increased DNA damage is a surrogate marker for the response of cells to this drug combination.     

Cloning of genetically tagged chromosome break sequences reveals new fragile sites at 6p21 and 13q22

Fragile sites are specific genomic loci that are especially prone to chromosome breakage. For the human genome there are 31 rare fragile sites and 88 common fragile sites listed in the National Center for Biotechnology Information database; however, the exact number remains unknown. In this study, unstable DNA sequences, which have been previously tagged with a marker gene, were cloned and provided starting points for the characterization of two aphidicolin inducible common fragile sites. Mapping of these unstable regions with six-color fluorescence in situ hybridization revealed two new fragile sites at 6p21 and 13q22, which encompass genomic regions of 9.3 and 3.1 Mb, respectively. According to the fragile site nomenclature they were consequently entitled as FRA6H and FRA13E. Both identified regions are known to be associated with recurrent aberrations in malignant and nonmalignant disorders. It is conceivable that these fragile sites result in genetic damage that might contribute to cancer phenotypes such as osteosarcoma, breast and prostate cancer.     

Integration of human papillomavirus 16 DNA and genomic rearrangements in immortalized human keratinocyte lines

Five foreskin-derived keratinocyte lines, immortalized by transfection of human papillomavirus (HPV16) DNA, were cytogenetically abnormal, exhibiting numerical deviations and altered chromosomes due to translocations, deletions, achromatic lesions, or partial duplications. Furthermore, all lines had cells with either homogeneously staining regions or double minute chromosomes, alterations associated with malignancy or drug resistance. None of these lines were tumorigenic in nude mice, showing that such alterations which are a manifestation of DNA amplification also occur in nonneoplastic cells. By in situ chromosome hybridization, viral sequences were identified on abnormal chromosomes at the junction of chromosome translocations, at achromatic lesions and within homogeneously staining regions and duplicated chromosome segments. Thus, for the first time in an experimental system, HPV16 integration into the cellular genome was associated with the induction of a subset of chromosome alterations. HPV16 integration that frequently occurred at fragile sites and near protooncogenes may be a critical alteration which confers a selective growth advantage and an indefinite proliferative potential to HPV-transfected cells.     

Low-frequency common fragile sites: link to neuropsychiatric disorders?

Common fragile sites are unstable chromosomal regions that predispose chromosomes to breakage and rearrangements. Recombinogenic DNA sequences encompassing these sites may contribute to both germinal and somatic genomic mutations, and the genomic instability at these regions might cause severe inherited disorders or predispose to cancer. In this review, we discuss the characterization of common fragile site FRA13A within the neurobeachin gene, which is involved in development and function of the central nervous system. We raise the possibility of an implication of common fragile sites in neuropsychiatric disorders and overview previous and recent reports concerning individual variability of expression of common fragile sites in human populations.     

p53, CHK2, and CHK1 genes in Finnish families with Li-Fraumeni syndrome: further evidence of CHK2 in inherited cancer predisposition

Germ-line mutations in the p53 gene predispose individuals to Li-Fraumeni syndrome (LFS). The cell cycle checkpoint kinases CHK1 and CHK2 act upstream of p53 in DNA damage responses, and recently rare germ-line mutations in CHK2 were reported in LFS families. We have analyzed CHK1, CHK2, and p53 genes for mutations in 44 Finnish families with LFS, Li-Fraumeni-like syndrome, or families phenotypically suggestive of LFS with conformation-sensitive gel electrophoresis. Five different disease-causing mutations were observed in 7 families (7 of 44 families; 15.9%): 4 in the p53 gene (5 of 44 families; 11.4%) and 1 in the CHK2 gene (2 of 44 families; 4.5%). Interestingly, the other CHK2-mutation carrier also has a mutation in the MSH6 gene. The cancer phenotype in the CHK2-families was not characteristic of LFS, and may indicate variable phenotypic expression in the rare families with CHK2 mutations. No mutations in the CHK1 gene were identified. Additional work is necessary to completely unravel the molecular background of LFS.     

Chromosomal fragile site FRA16D and DNA instability in cancer

It has been proposed that common aphidicolin-inducible fragile sites, in general, predispose to specific chromosomal breakage associated with deletion, amplification, and/or translocation in certain forms of cancer. Although this appears to be the case for the fragile site FRA3B and may be the case for FRA7G, it is not yet clear whether this association is a general property of this class of fragile site. The major aim of the present study was to determine whether the FRA16D chromosomal fragile site locus has a role to play in predisposing DNA sequences within and adjacent to the fragile site to DNA instability (such as deletion or translocation), which could lead to or be associated with neoplasia. We report the localization of FRA16D within a contig of cloned DNA and demonstrate that this fragile site coincides with a region of homozygous deletion in a gastric adenocarcinoma cell line and is bracketed by translocation breakpoints in multiple myeloma, as reported previously (Chesi, M., et al., Blood, 91: 4457-4463, 1998). Therefore, given similar findings at the FRA3B and FRA7G fragile sites, it is likely that common aphidicolin-inducible fragile sites exhibit the general property of localized DNA instability in cancer cells.     

Ku affects the ataxia and Rad 3-related/CHK1-dependent S phase checkpoint response after camptothecin treatment

Camptothecin (CPT) that targets DNA topoisomerase I is one of the most promising broad-spectrum anticancer drugs in development today. The cytotoxicity of CPT is S phase (S)-specific because the collision of advancing replication forks with CPT-topoisomerase I-DNA complexes results in DNA damage. After DNA damage, proliferating cells could actively slow down the DNA replication through an S checkpoint to provide time for repair. We report now that there is an activated S checkpoint response in CPT-treated mammalian cells. This response is regulated by Ataxia and Rad3-related (ATR)/CHK1 pathway. Compared with their wild-type counterparts, CPT-treated Ku80-/- cells showed stronger inhibition of DNA replication. This stronger inhibition had no relationship with DNA-dependent protein kinase (DNA-PK) activity but correlated with the higher activities of ATR and the higher activities of CHK1 in such cells. Not only caffeine, the nonspecific inhibitor of ATR, or UCN-01, the nonspecific inhibitor of CHK1, but also the specific CHK1 antisense oligonucleotide abolished the stronger inhibition of DNA replication in CPT-treated Ku80-/- cells. These results in aggregate indicated that the stronger S checkpoint in CPT-treated Ku80-/- cells is regulated through the highly activated ATR/CHK1 pathway.     

Characterization of a conserved aphidicolin-sensitive common fragile site at human 4q22 and mouse 6C1: possible association with an inherited disease and cancer

Fragile sites are classified as common or rare depending on their occurrence in the populations. While rare sites are mainly associated with inherited diseases, common sites have been involved in somatic rearrangements found in the chromosomes of cancer cells. Here we study a mouse locus containing the ionotropic glutamate receptor delta 2 (grid2) gene in which spontaneous chromosome rearrangements occur frequently, giving rise to mutant animals in inbred populations. We identify and clone common fragile sites overlapping the mouse grid2 gene and its human ortholog GRID2, lying respectively at bands 6C1 and 4q22 in a 7-Mb-long region of synteny. These results show a third example of orthologous common sites conserved at the molecular level, and reveal an unexpected link between an inherited disease and an aphidicolin-sensitive region. Recurrent deletions of subregions of band 4q22 have been previously described in human hepatocellular carcinomas. This 15-Mb-long region appears precisely centered on the site described here, which strongly suggests that it also plays a specific role in hepatic carcinogenesis.     

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

Objective

Overexpression of checkpoint kinase 1 (CHK1) is associated with poorer patient outcome and therapeutic resistance in multiple tumor models. Inhibition of CHK1 has been proposed as a strategy to increase the effectiveness of chemotherapeutic agents, especially in p53-deficient tumors. In this study, we evaluated the effects of a novel CHK1 inhibitor, MK-8776, in combination with pemetrexed (PMX) on cell proliferation and survival in a panel of p53 mutant non-small cell lung cancer (NSCLC) cell lines.

Methods

We examined CHK1 expression in 442 resected lung adenocarcinoma specimens using Affymetrix U133A gene expression arrays. We correlated CHK1 mRNA expression with patient survival, tumor differentiation and genomic complexity. We evaluated CHK1 levels in NSCLC cell lines and identified four p53 mutant cell lines with variable CHK1 expression (H1993, H23, H1437 and H1299) based on publicly available gene expression data. We confirmed differential CHK1 mRNA and CHK1 protein levels by qRT-PCR, ELISA, Western Blot analysis (WB) and immunohistochemistry. We examined cell line sensitization to PMX in response to CHK1 inhibition with MK-8776 using WST-1 and clonogenic survival assays.

Results

We found that elevated CHK1 expression in primary lung adenocarcinomas correlates with poor tumor differentiation and significantly worse patient survival. Tumors with elevated CHK1 mRNA levels have a higher number of gene mutations and DNA copy number gain or amplifications. CHK1 inhibition by MK-8776 enhances sensitivity of NSCLC cell lines to PMX. CHK1 mRNA and protein expression are variable among NSCLC cell lines, and cells expressing higher levels of CHK1 protein are more sensitive to the CHK1 inhibition by MK-8776 as compared to low CHK1 expressing cells.

Conclusions

These findings suggest that CHK1 levels may not only serve as a biomarker of poor prognosis in surgically-resected lung adenocarcinomas, but could also be a predictive marker for CHK1 inhibitor sensitivity, pending in vivo and clinical confirmation.     

The regulation of CHK2 in human cancer

Exceptional progress has been made in the past two decades in mapping oncogenes and tumour suppressors, defining a function for these master switches, and identifying novel anti-cancer drug targets. The p53 tumour suppressor is a central component of a DNA-damage-inducible pathway controlled by the ataxia telangiectasia mutated (ATM) and CHK2 protein kinases that have a central role in cancer suppression. One limitation of current human cancer research is the difficulty in developing genetic models that reveal the post-translational regulation of a growth suppressor like CHK2 within the microenvironment of a human tumour. Gaining such insights is important since yeast models and human tissue culture cell lines do not necessarily predict how enzymes like CHK2 are regulated in vivo, and therefore what factors can affect CHK2 tumour suppressor function. Translational cancer research aims to link basic research methodologies and clinical biology by uncovering cancer-specific pathways not revealed by other approaches. This approach is exemplified by two studies in this edition of Oncogene: both use a set of well-characterized human cancers with the objective of identifying novel post-translational control of the tumour suppressor CHK2. The authors have revealed two unexpected epigenetic modifications of the CHK2 pathway in vivo: (1) constitutive phosphorylation of CHK2 at its ATM-activated site in the absence of exogenous DNA damage; and (2) the production of hyper-spliced and inactive isoforms of CHK2. These studies highlight the need to develop model systems to understand why CHK2-activating pathways are being triggered or suppressed in different human cancers and whether the splicing machinery can be manipulated to control the activity of CHK2 for therapeutic benefit.     

Targeting CHK1 inhibits cell proliferation in FLT3-ITD positive acute myeloid leukemia

CHK1 Ser/Thr kinase, a well characterized regulator of DNA damage response, is also involved in normal cell cycle progression. In this study, we investigate how CHK1 participates to proliferation of acute myeloid leukemia cells expressing the mutated FLT3-ITD tyrosine kinase receptor. Pharmacological inhibition of CHK1 as well as its shRNA mediated down regulation reduced the proliferation rate of FLT-ITD expressing leukemic cell lines in a cytostatic manner. Flow cytometry analysis revealed no accumulation in a specific phase of the cell cycle upon CHK1 inhibition. Accordingly, lentiviral-mediated CHK1 overexpression increased the proliferation rate of FLT3-ITD expressing cells, as judged by cell viability and [3H] thymidine incorporation experiments. By contrast, expression of a ser280 mutant did not, suggesting that phosphorylation of this residue is an important determinant of CHK1 proliferative function. Clonogenic growth of primary leukemic cells from patients in semi-solid medium was reduced upon CHK1 inhibition, confirming the data obtained with leukemic established cell lines. Surprisingly, 3 out of 4 CHK1 inhibitory compounds tested in this study were also potent inhibitors of the FLT3-ITD tyrosine kinase receptor. Altogether, these data identify CHK1 as a regulator of FLT3-ITD-positive leukemic cells proliferation, and they open interesting perspectives in terms of new therapeutic strategies for these pathologies.